Bioscience Reports: this is an Accepted Manuscript, not the final Version of Record. You are
encouraged to use the Version of Record that, when published, will replace this version. The most
up-to-date version is available at http://dx.doi.org/10.1042/BSR20160364. Please cite using the DOI
10.1042/BSR20160364
BIOSCIENCE REPORTS
ACCEPTED MANUSCRIPT
The DnaE Polymerase from Deinococcus radiodurans features RecA‐ dependent DNA Polymerase activity Lorenzo Randi, Alessandro Perrone, Mirko Maturi, Fabrizio Dal Piaz, Michela Camerani, Alejandro Hochkoeppler Cite as Bioscience Reports (2016) DOI: 10.1042/BSR20160364
Copyright 2016 The Author(s).
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published, will replace this manuscript and be freely available under a Creative Commons licence.
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http://www.portlandpresspublishing.com/content/open-access-policy#Archiving).
ACCEPTED MANUSCRIPT
We report here on the catalytic properties of the Deinococcus radiodurans DNA Polymerase III α subunit (αDr). The αDr enzyme was overexpressed in E. coli, both in soluble form and as inclusion bodies. When purified from soluble protein extracts, αDr was found to be tightly associated with E. coli RNA Polymerase, from which αDr could not be dissociated. On the contrary, when refolded from inclusion bodies, αDr was devoid of E. coli RNA Polymerase, and was purified to homogeneity. When assayed with different DNA substrates, αDr featured slower DNA extension rates when compared to the corresponding enzyme from E. coli (αEc), unless under high ionic strength conditions or in the presence of manganese. Further assays were performed using a single‐stranded and a double‐stranded DNA, whose recombination yields a DNA substrate. Surprisingly, αDr was found to be incapable of recombination‐dependent DNA polymerase activity, while αEc was competent in this action. However, in the presence of the RecA recombinase αDr was able to efficiently extend the DNA substrate produced by recombination. Upon comparing the rates of RecA‐dependent and RecA‐independent DNA polymerase activities, we detected a significant activation of αDr by the recombinase. Conversely, the activity of αEc was found maximal under non‐recombination conditions. Overall, our observations indicate a sharp contrast between the catalytic actions of αDr and αEc, with αDr more performing under recombination conditions, and αEc preferring DNA substrates whose extension does not require recombination events. TheDnaEPolymerasefromDeinococcusradioduransfeaturesRecAǦ
dependentDNAPolymeraseactivity
‘”‡œ‘ƒ†‹ȗǡŽ‡••ƒ†”‘‡””‘‡ȗǡ‹”‘ƒ–—”‹ȗǡƒ„”‹œ‹‘ƒŽ‹ƒœȚǡ‹…Š‡Žƒ
ƒ‡”ƒ‹ȗǡŽ‡Œƒ†”‘‘…Š‘‡’’Ž‡”ȗι͉
ȗ‡’ƒ”–‡– ‘ˆ Šƒ”ƒ…› ƒ† ‹‘–‡…Š‘Ž‘‰›ǡ ‹˜‡”•‹–› ‘ˆ ‘Ž‘‰ƒǡ ‹ƒŽ‡ ‹•‘”‰‹‡–‘ Ͷǡ
ͶͲͳ͵͸‘Ž‘‰ƒȋ–ƒŽ›Ȍ
Ț‡’ƒ”–‡–‘ˆ‡†‹…‹‡ǡ‹˜‡”•‹–›‘ˆƒŽ‡”‘ǡ‹ƒ
‹‘˜ƒ‹ƒ‘Ž‘ͳ͵ʹǡͺͶͲͺͶ‹•…‹ƒ‘
ȋ–ƒŽ›Ȍ
ι
ǡ‹˜‡”•‹–›‘ˆ‹”‡œ‡ǡ‹ƒ†‡ŽŽƒƒ•–”—……‹ƒ͵ǡͷͲͲͳͻ‡•–‘‹‘”‡–‹‘ȋ–ƒŽ›Ȍ
͉‘™Š‘…‘””‡•’‘†‡…‡•Š‘—Ž†„‡ƒ††”‡••‡†ǣ
”‘ˆǤŽ‡Œƒ†”‘‘…Š‘‡’’Ž‡”
‡’ƒ”–‡–‘ˆŠƒ”ƒ…›ƒ†‹‘–‡…Š‘Ž‘‰›
‹˜‡”•‹–›‘ˆ‘Ž‘‰ƒ
‹ƒŽ‡‹•‘”‰‹‡–‘Ͷ
ͶͲͳ͵͸‘Ž‘‰ƒ
–ƒŽ›
Š‘‡ǣΪΪ͵ͻͲͷͳʹͲͻ͵͸͹ͳ
ƒšǣΪΪ͵ͻͲͷͳʹͲͻ͵͸͹͵
Ǧƒ‹ŽǣƒǤŠ‘…Š‘‡’’Ž‡”̷—‹„‘Ǥ‹–
Keywords:DeinococcusradioduransǢƒ‘Ž›‡”ƒ•‡Ǣ‘Ž›‡”ƒ•‡ǢȽ•—„—‹–Ǣ
‡…ǢEscherichiacoliǤ
SummaryStatement:
‡ ”‡’‘”– Š‡”‡ ‘ –Š‡ …ƒ–ƒŽ›–‹… ’”‘’‡”–‹‡• ‘ˆ Deinococcus radiodurans ƒ ‘Ž›‡”ƒ•‡ǡ
™Š‹…Š ‡Ž‘‰ƒ–‹‘ ‡ˆˆ‹…‹‡…› ™ƒ• …‘’ƒ”‡† ™‹–Š –Š‡ Š‘‘Ž‘‰‘—• Escherichia coli
‘Ž›‡”ƒ•‡Ǥ‘–”ƒ”›–‘–Š‡Žƒ––‡”ǡ–Š‡†‡‹‘…‘……ƒŽ‡œ›‡™ƒ•ˆ‘—†–‘„‡•–”‹…–Ž›†‡’‡†‡–
‘‡…”‡…‘„‹ƒ•‡Ǥ
ShortTitle:Deinococcusradioduransƒ‘Ž›‡”ƒ•‡‹•ƒ…–‹˜ƒ–‡†„›‡…”‡…‘„‹ƒ•‡
ABSTRACT
‡”‡’‘”–Š‡”‡‘–Š‡…ƒ–ƒŽ›–‹…’”‘’‡”–‹‡•‘ˆ–Š‡Deinococcusradiodurans‘Ž›‡”ƒ•‡
Ƚ •—„—‹– ȋȽ”ȌǤ Š‡ Ƚ” ‡œ›‡ ™ƒ• ‘˜‡”‡š’”‡••‡† ‹ E. coliǡ „‘–Š ‹ •‘Ž—„Ž‡ ˆ‘” ƒ† ƒ•
‹…Ž—•‹‘ „‘†‹‡•Ǥ Š‡ ’—”‹ˆ‹‡† ˆ”‘ •‘Ž—„Ž‡ ’”‘–‡‹ ‡š–”ƒ…–•ǡ Ƚ” ™ƒ• ˆ‘—† –‘ „‡ –‹‰Š–Ž›
ƒ••‘…‹ƒ–‡† ™‹–Š E. coli ‘Ž›‡”ƒ•‡ǡ ˆ”‘ ™Š‹…Š Ƚ” …‘—Ž† ‘– „‡ †‹••‘…‹ƒ–‡†Ǥ  –Š‡
…‘–”ƒ”›ǡ™Š‡”‡ˆ‘Ž†‡†ˆ”‘‹…Ž—•‹‘„‘†‹‡•ǡȽ”™ƒ•†‡˜‘‹†‘ˆE.coli‘Ž›‡”ƒ•‡ǡƒ†
™ƒ• ’—”‹ˆ‹‡† –‘ Š‘‘‰‡‡‹–›Ǥ Š‡ ƒ••ƒ›‡† ™‹–Š †‹ˆˆ‡”‡– •—„•–”ƒ–‡•ǡ Ƚ” ˆ‡ƒ–—”‡†
•Ž‘™‡”‡š–‡•‹‘”ƒ–‡•™Š‡…‘’ƒ”‡†–‘–Š‡…‘””‡•’‘†‹‰‡œ›‡ˆ”‘E.coliȋȽ…Ȍǡ
—Ž‡••—†‡”Š‹‰Š‹‘‹…•–”‡‰–Š…‘†‹–‹‘•‘”‹–Š‡’”‡•‡…‡‘ˆƒ‰ƒ‡•‡Ǥ—”–Š‡”ƒ••ƒ›•
™‡”‡ ’‡”ˆ‘”‡† —•‹‰ ƒ •‹‰Ž‡Ǧ•–”ƒ†‡† ƒ† ƒ †‘—„Ž‡Ǧ•–”ƒ†‡† ǡ ™Š‘•‡ ”‡…‘„‹ƒ–‹‘
›‹‡Ž†• ƒ •—„•–”ƒ–‡Ǥ —”’”‹•‹‰Ž›ǡ Ƚ” ™ƒ• ˆ‘—† –‘ „‡ ‹…ƒ’ƒ„Ž‡ ‘ˆ ”‡…‘„‹ƒ–‹‘Ǧ
†‡’‡†‡–’‘Ž›‡”ƒ•‡ƒ…–‹˜‹–›ǡ™Š‹Ž‡Ƚ…™ƒ•…‘’‡–‡–‹–Š‹•ƒ…–‹‘Ǥ‘™‡˜‡”ǡ‹–Š‡
’”‡•‡…‡ ‘ˆ –Š‡ ‡… ”‡…‘„‹ƒ•‡ Ƚ” ™ƒ• ƒ„Ž‡ –‘ ‡ˆˆ‹…‹‡–Ž› ‡š–‡† –Š‡ •—„•–”ƒ–‡
’”‘†—…‡† „› ”‡…‘„‹ƒ–‹‘Ǥ ’‘ …‘’ƒ”‹‰ –Š‡ ”ƒ–‡• ‘ˆ ‡…Ǧ†‡’‡†‡– ƒ† ‡…Ǧ
‹†‡’‡†‡– ’‘Ž›‡”ƒ•‡ ƒ…–‹˜‹–‹‡•ǡ ™‡ †‡–‡…–‡† ƒ •‹‰‹ˆ‹…ƒ– ƒ…–‹˜ƒ–‹‘ ‘ˆ Ƚ” „› –Š‡
”‡…‘„‹ƒ•‡Ǥ ‘˜‡”•‡Ž›ǡ –Š‡ ƒ…–‹˜‹–› ‘ˆ Ƚ… ™ƒ• ˆ‘—† ƒš‹ƒŽ —†‡” ‘Ǧ”‡…‘„‹ƒ–‹‘
…‘†‹–‹‘•Ǥ˜‡”ƒŽŽǡ‘—”‘„•‡”˜ƒ–‹‘•‹†‹…ƒ–‡ƒ•Šƒ”’…‘–”ƒ•–„‡–™‡‡–Š‡…ƒ–ƒŽ›–‹…ƒ…–‹‘•
‘ˆ Ƚ” ƒ† Ƚ…ǡ ™‹–Š Ƚ” ‘”‡ ’‡”ˆ‘”‹‰ —†‡” ”‡…‘„‹ƒ–‹‘ …‘†‹–‹‘•ǡ ƒ† Ƚ…
’”‡ˆ‡””‹‰•—„•–”ƒ–‡•™Š‘•‡‡š–‡•‹‘†‘‡•‘–”‡“—‹”‡”‡…‘„‹ƒ–‹‘‡˜‡–•Ǥ
ʹ
INTRODUCTION
‘Ž›‡”ƒ•‡• ƒ”‡ ’‡…—Ž‹ƒ” ‡œ›‡•ǡ ˆ‡ƒ–—”‹‰ ƒ …‘•‡”˜‡† —Ž–‹†‘ƒ‹ ‘Ž‡…—Žƒ”
ƒ”…Š‹–‡…–—”‡Ǥ  ’ƒ”–‹…—Žƒ”ǡ –Š‡ …‘ˆ‘”ƒ–‹‘ ‘ˆ ‘Ž›‡”ƒ•‡• ȋ ‘Ž•Ȍ ”‡•‡„Ž‡• ƒ
‘’‡”‹‰Š–ǦŠƒ†ǡƒ†…‘–ƒ‹•–Š”‡‡ƒ‹†‘ƒ‹•ǡ†‡‘–‡†–Š—„ǡ’ƒŽǡƒ†ˆ‹‰‡”•ȏͳȐǤŠ‡
–Š—„ †‘ƒ‹ –‹‰Š–Ž› ƒ••‘…‹ƒ–‡• –‘ –Š‡ †‘—„Ž‡Ǧ•–”ƒ†‡† ’‘”–‹‘ ‘ˆ –Š‡ •—„•–”ƒ–‡ǡ
™Š‡”‡ƒ•–Š‡͵ǯǦ‡†‘ˆ–Š‡’”‹‡”ƒ†–Š‡•‹‰Ž‡Ǧ•–”ƒ†‡†–‡’Žƒ–‡ƒ”‡ƒŽŽ‘…ƒ–‡†‹
–Š‡ ’ƒŽ ƒ† ‹ –Š‡ ˆ‹‰‡”• †‘ƒ‹ǡ ”‡•’‡…–‹˜‡Ž› ȏʹȐǤ Š‡ •—„•‡“—‡– „‹†‹‰ ‘ˆ ƒ
†‡‘š›—…Ž‡‘•‹†‡Ǧ–”‹’Š‘•’Šƒ–‡ȋ†Ȍ–”‹‰‰‡”•–Š‡‘˜‡‡–‘ˆ–Š‡ˆ‹‰‡”•–‘™ƒ”†•–Š‡’ƒŽǡ
’‘‹•‹‰ –Š‡ ƒ…–‹˜‡ •‹–‡ –‘ ’”‘ˆ‹…‹‡– …ƒ–ƒŽ›•‹•ǡ ‹Ǥ‡Ǥ –Š‡ —…Ž‡‘’Š‹Ž‹… ƒ––ƒ… „› –Š‡ ͵ǯǦ ‘ˆ –Š‡
’”‹‡”–‘–Š‡ȽǦ’Š‘•’Šƒ–‡‘ˆ–Š‡†ȏ͵ȐǤ‡•‹†‡•–Š‹•’”‹ƒ”›‡š–‡•‹‘ƒ…–‹˜‹–›ǡ–Š‡
…‘’Ž‡š–ƒ•‘ˆ‰‡‘‡”‡’Ž‹…ƒ–‹‘†‡ƒ†•–‘‘Ž•–Š‡‡š‡”–‹‘‘ˆ“—‹–‡ƒ—„‡”‘ˆ
•‡…‘†ƒ”›‡••‡–‹ƒŽˆ—…–‹‘•Ǥ‘‰–Š‡•‡ˆ—…–‹‘•ǡŠ‹‰Šˆ‹†‡Ž‹–›ƒ†’”‘…‡••‹˜‡”‡’Ž‹…ƒ–‹‘ǡ
ƒ•™‡ŽŽƒ•’”‘’‡”…‘‘”†‹ƒ–‹‘‘ˆŽ‡ƒ†‹‰ƒ†Žƒ‰‰‹‰•–”ƒ†‡š–‡•‹‘ǡƒ”‡ƒ……‘’Ž‹•Š‡†
„›‘ŽŠ‘Ž‘‡œ›‡•…‘’‘•‡†‘ˆ†‹ˆˆ‡”‡–•—„—‹–•ǡ–Š‡ƒ••‡„Ž›‘ˆ™Š‹…Š†‡–‡”‹‡•
–Š‡ƒ…–—ƒŽ‡œ›‡ƒ…–‹˜‹–‹‡•ȏʹȐǤEscherichiacoli†‘‡•‡š’”‡••ˆ‹˜‡†‹•–‹…–‘Ž•ǡ™‹–Š
‘Ž”‡•’‘•‹„Ž‡ˆ‘”‰‡‘‡”‡’Ž‹…ƒ–‹‘ȏͶǦ͸Ȑǡ‘Ž‡••‡–‹ƒŽˆ‘””‡‘˜‹‰–Š‡—Ž–‹’Ž‡
’”‹‡”• ‡…‡••ƒ”› ˆ‘” Žƒ‰‰‹‰ •–”ƒ† ”‡’Ž‹…ƒ–‹‘ ȏ͹ǡͺȐǡ ƒ† ‘Ž• ǡ ǡ ƒ† ȏͻǦͳʹȐǡ
‡˜‡–—ƒŽŽ› ‡‰ƒ‰‡† ‹ –Š‡ ”‡’Ž‹…ƒ–‹‘ ‘ˆ †ƒƒ‰‡† Ǥ E. coli ‘Ž ‹• …‘’‘•‡† ‘ˆ ƒ
…ƒ–ƒŽ›–‹……‘”‡ǡƒƒ…‘’Ž‡šǡƒ†ƒȾǦ…Žƒ’ǤŠ‡…ƒ–ƒŽ›–‹……‘”‡†‘‡•…‘–ƒ‹–Š‡Ƚǡɂǡƒ†Ʌ
•—„—‹–• ȏͳ͵Ȑǡ ”‡•’‡…–‹˜‡Ž› ˆ‡ƒ–—”‹‰ ͷǯǦ͵ǯ ’‘Ž›‡”ƒ•‡ ȏͳͶȐǡ ͵ǯǦͷǯ ‡š‘—…Ž‡ƒ•‡ ȋ’”‘‘ˆ”‡ƒ†‹‰Ȍ
ȏͳͷȐǡƒ†ɂǦ•–ƒ„‹Ž‹œ‹‰ƒ…–‹˜‹–›ȏͳ͸ǡͳ͹ȐǤ‘–ƒ„Ž›ǡ‘ŽȽ•—„—‹–ǡ™Š‹…Š‹•…‘†‡†„›–Š‡
dnaE‰‡‡ǡ‹•–Š‡‘Ž›‡••‡–‹ƒŽ”‡’Ž‹…ƒ•‡‹E.coliǤŠ‡ƒ…‘’Ž‡š…‘–ƒ‹•–Š‡ɒǡɁǡɁǯǡɖǡ
ƒ† ɗ •—„—‹–• ȏͳͺȐǡ ƒ……‘”†‹‰ –‘ ƒ •–‘‹…Š‹‘‡–”› ɒ͵ɁɁǯɖɗ ȏͳͻȐǤ Š‹• …‘’Ž‡š …ƒ–ƒŽ›œ‡• –Š‡
Ž‘ƒ†‹‰ ‘ˆ –Š‡ ȾǦ…Žƒ’ ‘–‘ ǡ ƒ† ‹–‡”ƒ…–• ™‹–Š –Š‡ …‘”‡ Ƚ •—„—‹–Ǥ Š‡ ȾǦ…Žƒ’ ‹• ƒ
͵
Š‘‘†‹‡”ǡƒ†…‘ˆ‡”•–‘–Š‡Š‘Ž‘‡œ›‡Š‹‰Š’”‘…‡••‹˜‹–›„›–‡–Š‡”‹‰–Š‡‘Ž›‡”ƒ•‡–‘
–Š‡•—„•–”ƒ–‡ȏʹͲǡʹͳȐǤƒ††‹–‹‘ǡ–Š‡’”‘‰”‡••‹‘‘ˆƒ”‡’Ž‹…ƒ–‹‘ˆ‘”‹•ƒ••‹•–‡†„›–Š‡
ƒ••‘…‹ƒ–‹‘‘ˆ‘Ž™‹–ŠŠ‡Ž‹…ƒ•‡ƒ†’”‹ƒ•‡ȏʹʹǡʹ͵ȐǤ–‡”‡•–‹‰Ž›ǡ‹–™ƒ••Š‘™
–Šƒ–ȾǦ…Žƒ’ǡŠ‡Ž‹…ƒ•‡ǡƒ†’”‹ƒ•‡ƒ”‡…‘’‡–‡–‹‹–‡”ƒ…–‹‰™‹–ŠE.coli‘Ž•ǡǡ
ƒ†ȏʹͶȐǡ™Š‘•‡ƒ…–‹‘‹•ƒ••‹•–‡†„›–Š‡‡…”‡…‘„‹ƒ•‡ȏʹͷǦʹ͹ȐǤ‘”‡”‡…‡–Ž›ǡ‹–™ƒ•
ƒŽ•‘‡Ž‡‰ƒ–Ž›†‡‘•–”ƒ–‡†–Šƒ–‡…ƒ…–‹˜ƒ–‡•”‡’Ž‹•‘‡•…‘–ƒ‹‹‰‘Žǡ‘Žǡ‘”
‘Ž ȏʹͺȐǡ ƒ† ‹Š‹„‹–• ‘Ž Š‘Ž‘‡œ›‡ ȏʹͺȐǤ  ƒ††‹–‹‘ǡ ‹– ™ƒ• ‘„•‡”˜‡† –Šƒ– –Š‡
‘˜‡”‡š’”‡••‹‘ ‘ˆ ‘Ž ƒ† ‡… •Ž‘™• †‘™ –Š‡ ’”‘‰”‡••‹‘ ‘ˆ ‘Ž ”‡’Ž‹…ƒ–‹‘ ˆ‘”•
ȏʹͻȐǡ•—‰‰‡•–‹‰–Šƒ–‡…‹Š‹„‹–•–Š‡ƒ…–‹‘‘ˆ‘ŽŠ‘Ž‘‡œ›‡‹E.coliǤ
Deinococcusradiodurans‹•ƒ
”ƒǦ’‘•‹–‹˜‡„ƒ…–‡”‹—ǡ„‡Ž‘‰‹‰–‘–Š‡‡‹‘…‘……ƒŽ‡•‘”†‡”ǡ
™Š‘•‡ ‡„‡”• ˆ‡ƒ–—”‡ ‘—–•–ƒ†‹‰ ”ƒ†‹‘”‡•‹•–ƒ…‡ ȏ͵ͲȐǤ Deinococcus radiodurans ™ƒ•
‹†‡‡†ˆ‹”•–‹•‘Žƒ–‡†ˆ”‘…ƒ‡†‡ƒ–•ƒ’Ž‡•ǡ‡š’‘•‡†–‘ɀǦ”ƒ›•‹‘”†‡”–‘‘„–ƒ‹ƒ•–‡”‹Ž‡ǡ
ƒ† Š‡…‡ •–ƒ„Ž‡ǡ ˆ‘‘† •—’’Ž› ȏ͵ͳȐǤ ‘–”ƒ”› –‘ –Š‡ ˜ƒ•– ƒŒ‘”‹–› ‘ˆ ’”‘ƒ”›‘–‡•ǡ D.
radiodurans…‡ŽŽ•ƒ”‡’‘Ž›’Ž‘‹†ǡ™‹–Š–Š‡ƒ…–—ƒŽ’Ž‘‹†›—„‡”„‡‹‰ƒˆˆ‡…–‡†„›‰”‘™–Š’Šƒ•‡
ȏ͵ʹȐƒ†…—Ž–—”‡‡†‹—ȏ͵͵ȐǤƒ…Š‰‡‘‡…‘’›…‘•‹•–•‘ˆ–™‘ …Š”‘‘•‘‡•ȋ…‘–ƒ‹‹‰
ʹǤ͸ ƒ† ͲǤͶ „’Ȍ ƒ† –™‘ ’Žƒ•‹†•ǡ ˆ‡ƒ–—”‹‰ ͳ͹͹yͳͲ͵ ƒ† ͶͷǤ͹yͳͲ͵ „’ǡ ”‡•’‡…–‹˜‡Ž› ȏ͵ͶȐǤ
Š”‡‡†‹ˆˆ‡”‡–‘Ž•™‡”‡‹†‡–‹ˆ‹‡†‹D.radioduransǡ‹Ǥ‡Ǥ‘Žȏ͵ͷȐǡȏ͵͸Ȑǡƒ†
ȏ͵͹Ȑǡ ƒ‘‰ ™Š‹…Š ‘Ž Ƚ •—„—‹– ȋƒȌ ‹• ‡••‡–‹ƒŽ ˆ‘” ˜‹ƒ„‹Ž‹–› ȏ͵͸ǡ͵ͺȐǤ ƒ–Š‡”
ƒ•–‘‹•Š‹‰Ž›ǡ …‡ŽŽ• ‘ˆ D. radiodurans ‡š’‘•‡† –‘ ͳͶ 
›ǡ ƒ† …‘–ƒ‹‹‰ ˆ”ƒ‰‡–‡†
…Š”‘‘•‘‡•ǡ ƒ”‡ ƒ„Ž‡ –‘ ”‡…‘•–”—…– –Š‡‹” ‰‡‘‡• ™‹–Š‹ ͸Ǧ͹ Š ƒˆ–‡” ”ƒ†‹ƒ–‹‘ ‡š’‘•—”‡
ȏ͵ͲȐǤŠ‹Ž‡’‘Ž›’Ž‘‹†›‹•ƒ‘„˜‹‘—•”‡“—‹•‹–‡ˆ‘”–Š‹•‰‡‘‡”‡…‘•–”—…–‹‘…‘’‡–‡…‡ǡD.
radiodurans†‘‡•ƒŽ•‘ˆ‡ƒ–—”‡ƒ††‹–‹‘ƒŽƒ†’‡…—Ž‹ƒ”„‹‘…Š‡‹…ƒŽ’”‘’‡”–‹‡•”‡•’‘•‹„Ž‡ˆ‘”
‰‡‘‡‹–‡‰”‹–›ƒ‹–‡ƒ…‡Ǥ’ƒ”–‹…—Žƒ”ǡ™Š‡…‘•‹†‡”‹‰–Šƒ–‹‘‹œ‹‰”ƒ†‹ƒ–‹‘•‹†—…‡
•‡˜‡”‡ ‘š‹†ƒ–‹˜‡ •–”‡••ǡ ‹– ™ƒ• ”‡ƒŽ‹œ‡† –Šƒ– –Š‡ ”ƒ†‹ƒ–‹‘Ǧ”‡•‹•–ƒ…‡ ‘ˆ D. radiodurans ‹•
ƒ‹Ž›†—‡–‘„‹‘…Š‡‹…ƒŽˆƒ…–‘”•’”‡•‡”˜‹‰–Š‡’”‘–‡‘‡‘ˆ–Š‹•„ƒ…–‡”‹—ˆ”‘‘š‹†ƒ–‹‘
Ͷ
†ƒƒ‰‡• ȏ͵ͲȐǤ ‡˜‡”–Š‡Ž‡••ǡ –Š‡ …ƒ’ƒ„‹Ž‹–› ‘ˆ D. radiodurans –‘ ”‡…‘•–”—…– ‹–• Š‹‰ŠŽ›
ˆ”ƒ‰‡–‡† ‰‡‘‡ †‘‡• ƒŽ•‘ ‹’Ž› ƒ ‘—–•ƒ†‹‰ ”‡’ƒ‹” …‘’‡–‡…‡Ǥ ‘ ƒ……‘’Ž‹•Š
–Š‹• –ƒ•ǡ D. radiodurans ˆ‹”•– †‡’‡†• ‘ ‡…ǡ ™Š‹…Š ‹• ‡••‡–‹ƒŽ ˆ‘” ˜‹ƒ„‹Ž‹–› ȏ͵ͻȐ ƒ†
ˆ‡ƒ–—”‡• ͷǯǦ͵ǯ ‡š‘—…Ž‡ƒ•‡ ƒ…–‹˜‹–›Ǥ Š‡ ƒ…–‹‘ ‘ˆ –Š‹• ‡œ›‡ ’”‘†—…‡• ͵ǯ ‘˜‡”Šƒ‰• ƒ– –Š‡
‡š’‡•‡‘ˆ…Š”‘‘•‘ƒŽȀ’Žƒ•‹†ˆ”ƒ‰‡–•ǡ–”‹‰‰‡”‹‰–Š‡‡…Ǧ‡†‹ƒ–‡†Ž‘ƒ†‹‰‘ˆ‡…
‘–‘Ǥ—„•‡“—‡–Ž›ǡ–Š‡”‡…‘„‹ƒ•‡ƒ…–‹˜‹–›‘ˆ‡…ƒ†–Š‡’‘Ž›‡”ƒ•‡ƒ…–‹˜‹–›‘ˆ
ƒ…ƒ–ƒŽ›œ‡–Š‡”‡…‘„‹ƒ–‹‘‘ˆ‘˜‡”Žƒ’’‹‰Š‘‘Ž‘‰‘—•ˆ”ƒ‰‡–•ƒ†–Š‡‡š–‡•‹‘‘ˆ
–Š‡ ”‡…‘„‹ƒ– ‘Ž‡…—Ž‡•ǡ ”‡•’‡…–‹˜‡Ž›Ǥ Š‹• ‡…Šƒ‹•ǡ †‡‘‹ƒ–‡† ȋš–‡•‹˜‡
›–Š‡•‹•Ǧ‡’‡†‡– –”ƒ† ‡ƒŽ‹‰Ȍǡ †‘‡• ’”‘‰”‡••‹˜‡Ž› ‹…”‡ƒ•‡ –Š‡ ‘Ž‡…—Žƒ” ƒ•• ‘ˆ
ˆ”ƒ‰‡–•ǡ™Š‘•‡—„‡”†‡…”‡ƒ•‡•‹’ƒ”ƒŽŽ‡ŽǤ‡•‹†‡•–Š‡‹’ƒ‹”‡‡–‘ˆǡ„‘–Š
recA ƒ† dnaE —–ƒ–‹‘• …‘ˆ‡” ‹–‡”‡•–‹‰ ’Š‡‘–›’‡• –‘ D. radioduransǣ ‹Ȍ ȟrecA •–”ƒ‹•
ˆ‡ƒ–—”‡ ’‘‘” ˜‹ƒ„‹Ž‹–› ƒ† ‡š–”‡‡ ”ƒ†‹ƒ–‹‘ •‡•‹–‹˜‹–› ȏ͵ͻǦͶͳȐǢ ‹‹Ȍ —†‡” ’Š›•‹‘Ž‘‰‹…ƒŽ
…‘†‹–‹‘•ȋ‘‹””ƒ†‹ƒ–‹‘ȌǡrecA—–ƒ–•†‘”‡’Ž‹…ƒ–‡ƒ–•Ž‘™‡””ƒ–‡•™Š‡…‘’ƒ”‡†–‘
™‹Ž†Ǧ–›’‡ ȏ͵͸ȐǢ ‹‹‹Ȍ –‡’‡”ƒ–—”‡Ǧ•‡•‹–‹˜‡ dnaE —–ƒ–• ˆ‡ƒ–—”‡ Ž‘™‡” ”ƒ†‹ƒ–‹‘Ǧ”‡•‹•–ƒ…‡
–Šƒ •–”ƒ‹• †‡ˆ‡…–‹˜‡ ‹ ‘Ž›‡”ƒ•‡ ȋ‘ŽȌ ȏ͵ͷǡ͵͸ȐǤ  ’ƒ”–‹…—Žƒ”ǡ –Š‡ ’‘‘” ”‡’Ž‹…ƒ–‹‘ ‡ˆˆ‹…‹‡…› ‘ˆ —‹””ƒ†‹ƒ–‡† ‡…Ǧ •–”ƒ‹• •—‰‰‡•–• ƒ ”‘Ž‡ ˆ‘” –Š‹• ”‡…‘„‹ƒ•‡ ‹
ƒǦ…ƒ–ƒŽ›œ‡†”‡’Ž‹…ƒ–‹‘Ǥ
‘–Š ‘Ž ƒ† ‘Ž ˆ”‘ D. radiodurans ™‡”‡ ’”‡˜‹‘—•Ž› ’—”‹ˆ‹‡†ǡ ƒ† –Š‡‹”
…ƒ–ƒŽ›–‹…’‡”ˆ‘”ƒ…‡•™‡”‡‹˜‡•–‹‰ƒ–‡†ȏ͵ͷǡͶʹǦͶͷȐǤ–Š‡…‘–”ƒ”›ǡ–Š‡Ƚ•—„—‹–ȋƒȌ‘ˆ
D.radiodurans‘ŽȋŠ‡”‡†‘‹‹ƒ–‡†Ƚ”ȌŠƒ•‡˜‡”„‡‡‹•‘Žƒ–‡†–‘Š‘‘‰‡‹‹–›in
vitroǤ ‡”‡ ™‡ ”‡’‘”–‘ –Š‡ ‘˜‡”‡š’”‡••‹‘ƒ† ’—”‹ˆ‹…ƒ–‹‘ ‘ˆ–Š‹• ‡œ›‡ǡ ƒŽ‘‰ ™‹–Š –Š‡
…Šƒ”ƒ…–‡”‹œƒ–‹‘‘ˆ‹–•…ƒ–ƒŽ›–‹…’”‘’‡”–‹‡•ƒ––Š‡‡š’‡•‡‘ˆ†‹ˆˆ‡”‡–•—„•–”ƒ–‡•ǡ‹–Š‡
’”‡•‡…‡‘”‹–Š‡ƒ„•‡…‡‘ˆ‡…Ǥ—”–Š‡”ǡ™‡—•‡†–Š‡™‡ŽŽǦ‘™Ƚ•—„—‹–ȋƒȌ‘ˆE.
coli ‘Ž ȋŠ‡”‡ †‘‘‹ƒ–‡† Ƚ…Ȍ ƒ• ƒ ”‡ˆ‡”‡…‡ ’‘Ž›‡”ƒ•‡ǡ ƒ† ™‡ ”‡’‘”– ‘ –Š‡
…‘’ƒ”‹•‘‘ˆ–Š‡•‡–™‘‡œ›‡•ǡ™Š‘•‡ƒ…–‹‘‹•‡••‡–‹ƒŽˆ‘”–Š‡‹””‡•’‡…–‹˜‡‘”‰ƒ‹••Ǥ
ͷ
MATERIALSANDMETHODS
Bacterialstrains,plasmidsandgrowthmedia
Escherichia coli ʹͷͻͻ͵ ȋ‰‡‘–›’‡ǣ ȟȋaraDǦaraBȌ567ǡ rphǦ1ǡ ȟȋrhaDǦrhaBȌ568ǡ hsdR514Ȍ
™ƒ• ‘„–ƒ‹‡† „› ƒ”› ‡”Ž› ȋEscherichia coli ‡‡–‹… –‘… ‡–‡”ǡ ǡ ƒŽ‡ ‹˜‡”•‹–›Ȍǡ
ƒ† E. coli ͳͲ ȋ‰‡‘–›’‡ǣ Ǧ mcrA 'ȋmrrǦhsdRMSǦmcrBCȌ IͺͲlacZ'M15 'lacX74 recA1
araD139 'ȋaraǦleuȌ7697 galU galK rpsL endA1 nupGȌ ™ƒ• ’—”…Šƒ•‡† ˆ”‘ ˜‹–”‘‰‡
ȋƒ”Ž•„ƒ†ǡǡȌǤ‘–Š•–”ƒ‹•™‡”‡‰”‘™‹‡†‹—ȋͳͲǡͳͲǡƒ†ͷ‰Ȁǡ”‡•’‡…–‹˜‡Ž›ǡ
‘ˆ ”›’–‘‡ǡ ƒŽǡ ƒ† ‡ƒ•– š–”ƒ…–Ȍǡ •—’’Ž‡‡–‡†ǡ ™Š‡ ƒ’’”‘’”‹ƒ–‡ǡ ™‹–Š ͳͲͲ Ɋ‰Ȁ ‘ˆ
ƒ’‹…‹ŽŽ‹ǤŠ‡’Žƒ•‹†’ǦȽͳͳ͸Ͳǡ…‘–ƒ‹‹‰ƒ‹•‡”–…‘†‹‰ˆ‘”–Š‡ˆ—ŽŽǦŽ‡‰–ŠȽ•—„—‹–
‘ˆE.coli‘Ž›‡”ƒ•‡ȋȽ…Ȍǡ™ƒ•’”‡˜‹‘—•Ž›†‡•…”‹„‡†ȏͶ͸Ȑǡƒ†™‹ŽŽ„‡‹†‹…ƒ–‡†Š‡”‡
ƒ•’ǦȽ…ǤŠ‡‰‡‡…‘†‹‰ˆ‘”–Š‡ˆ—ŽŽǦŽ‡‰–ŠȽ•—„—‹–‘ˆ Deinococcusradiodurans ‘Ž›‡”ƒ•‡ ȋȽ”Ȍ ™ƒ• ‘’–‹‹œ‡† ˆ‘” E. coli …‘†‘ —•ƒ‰‡ ƒ† •›–Š‡•‹œ‡† „› ‡…”‹’–
ȋ‹•…ƒ–ƒ™ƒ›ǡǡȌǤŠ‡•›–Š‡–‹…‰‡‡™ƒ•‹•‡”–‡†‹–‘–Š‡’Ǧ‹•’Žƒ•‹†ǡ—•‹‰–Š‡
Ncoƒ†Pst•‹–‡•ƒ–ͷǯƒ†͵ǯ‡†•ǡ”‡•’‡…–‹˜‡Ž›ǡ›‹‡Ž†‹‰’ǦȽ”ǤŠ‹•˜‡…–‘”™ƒ•—•‡†–‘
–”ƒ•ˆ‘” „‘–Š E. coli •–”ƒ‹• ʹͷͻͻ͵ ƒ† ͳͲǡ –Š‡ –”ƒ•ˆ‘”ƒ–• ™‡”‡ ‹•‘Žƒ–‡† ƒ†
’—”‹ˆ‹‡†‘‡–”‹†‹•Š‡•…‘–ƒ‹‹‰ͳͲͲɊ‰Ȁ‘ˆƒ’‹…‹ŽŽ‹ǡƒ†–Š‡•–‘”‡†ƒ–ǦʹͲ鋐
Ǧ‰Ž›…‡”‘ŽȋͳͷΨǡ˜Ȁ˜Ȍ‡†‹—Ǥ”‘–‡‹•‘˜‡”‡š’”‡••‹‘™ƒ•–”‹‰‰‡”‡†‹‡†‹—„›–Š‡
ƒ††‹–‹‘‘ˆͳƒ”ƒ„‹‘•‡Ǥ
Proteinsoverexpression
Š‡‘˜‡”‡š’”‡••‹‘‘ˆȽ…™ƒ•’‡”ˆ‘”‡†ƒ•’”‡˜‹‘•—Ž›†‡•…”‹„‡†ȏͶ͹ȐǤŠ‡‘˜‡”‡š’”‡••‹‘
‘ˆ•‘Ž—„Ž‡Ƚ”™ƒ•ƒ––‡’–‡†—•‹‰„‘–ŠE.coli•–”ƒ‹•ͳͲƒ†ʹͷͻͻ͵ǤE.coliͳͲ
͸
–Š‡–ƒ”‰‡–’”‘–‡‹™ƒ•‡š’”‡••‡†ƒ–Ž‘™…‘…‡–”ƒ–‹‘•ǡ‹””‡•’‡…–‹˜‡Ž›‘ˆ–‡’‡”ƒ–—”‡‰”‘™–Š
ƒ†‹†—…–‹‘Ž‡‰–ŠǤ–Š‡…‘–”ƒ”›ǡ–Š‡ʹͷͻͻ͵•–”ƒ‹†‹†’‡”ˆ‘”—…Š„‡––‡”ǡƒ†™ƒ•
—•‡† –Š”‘—‰Š‘—– –‘ ‘˜‡”‡š’”‡•• •‘Ž—„Ž‡ Ƚ”Ǥ  ’ƒ”–‹…—Žƒ”ǡ •‹‰Ž‡ …‘Ž‘‹‡• ‘ˆ E. coli
ʹͷͻͻ͵Ȁ’ǦȽ” ™‡”‡ ’‹…‡† ˆ”‘ Ǧƒ’‹…‹ŽŽ‹ ‡–”‹ †‹•Š‡•ǡ –”ƒ•ˆ‡””‡† –‘ Ǧ
ƒ’‹…‹ŽŽ‹ Ž‹“—‹† ‡†‹—ǡ ƒ† ’”‡Ǧ…—Ž–—”‡† ƒ– ͵͹ ι ˆ‘” ͺ ŠǤ Š‡ ’”‡Ǧ…—Ž–—”‡• ™‡”‡ –Š‡
†‹Ž—–‡† ȋͳǣͷͲͲȌ ‹ ˆ”‡•Š ‡†‹—ǡ ‰”‘™ ˆ‘” ͳͷ Š ƒ– ͵͹ ιǡ ƒ† –Š‡ …—Ž–—”‡• ™‡”‡ ˆ‹ƒŽŽ›
‹†—…‡†ƒ–͵͹ιˆ‘”͵ŠǤ‡ŽŽ•™‡”‡…‘ŽŽ‡…–‡†„›…‡–”‹ˆ—‰ƒ–‹‘ȋͳͲǡͲͲͲš‰ǡʹͲ‹ǡͶιȌǡƒ†
–Š‡’‡ŽŽ‡–•™‡”‡•–‘”‡†ƒ–ǦʹͲ闐–‹Ž—•‡†Ǥ
‘‘˜‡”‡š’”‡••Ƚ”‹–Š‡ˆ‘”‘ˆ‹…Ž—•‹‘„‘†‹‡•ǡ•‹‰Ž‡…‘Ž‘‹‡•‘ˆE.coliͳͲȀ’ǦȽ”
™‡”‡’”‡Ǧ…—Ž–—”‡†‹‡†‹—ˆ‘”ͳͷŠƒ–͵͹ιǤŠ‡’”‡Ǧ…—Ž–—”‡•™‡”‡–Š‡†‹Ž—–‡†ȋͳǣͷͲͲȌ
‹ ˆ”‡•Š ‡†‹—ǡ ‰”‘™ ˆ‘” ͺ Šǡ ƒ† ˆ‹ƒŽŽ› ‹†—…‡† ˆ‘” ͳͷ Š ƒ– ͵͹ ιǤ ˆ–‡” Šƒ”˜‡•–ǡ –Š‡
…‡ŽŽ•’‡ŽŽ‡–•™‡”‡•–‘”‡†ƒ–ǦʹͲιǤ
Extractionofsolubleproteins
‘‹•‘Žƒ–‡•‘Ž—„Ž‡’”‘–‡‹‡š–”ƒ…–•ǡ…‡ŽŽ•™‡”‡”‡•—•’‡†‡†‹—ˆˆ‡”ǡ…‘•‹•–‹‰‘ˆͷͲ
”‹•ǦŽȋ’ͺȌǡͷͲƒŽǡͳǡʹǤͷǡƒ†‰‡–Ž›Š‘‘‰‡‹œ‡†™‹–Šƒ…‘Ž†
‰Žƒ••’‘––‡”ǤŠ‡…‡ŽŽ••—•’‡•‹‘™ƒ•–Š‡•—’’Ž‡‡–‡†™‹–Šͳ’Š‡›ŽǦ‡–Š›ŽǦ•—Žˆ‘›Ž
ˆŽ—‘”‹†‡ȋȌƒ†•—„Œ‡…–‡†–‘͹…›…Ž‡•‘ˆ•‘‹…ƒ–‹‘ƒ–͸Ǥƒ…Š•‘‹…ƒ–‹‘…›…Ž‡…‘•‹•–‡†
‘ˆͳͷ•‘ˆ‹’—Ž•‡ǡˆ‘ŽŽ‘™‡†„›ͳͷ•‘ˆ…‘‘Ž‹‰‹–‡”˜ƒŽǡˆ‘”ƒ–‘–ƒŽ–‹‡‘ˆʹ‹ǤŠ‡‡š–”ƒ…–•
ƒ……‘”†‹‰Ž›‘„–ƒ‹‡†™‡”‡…‡–”‹ˆ—‰‡†ȋͳͲǡͲͲͲš‰ǡʹͲ‹ǡͶιȌǡ–Š‡’‡ŽŽ‡–•™‡”‡†‹•…ƒ”†‡†ǡ
ƒ†–Š‡•—’‡”ƒ–ƒ–™ƒ•‹‡†‹ƒ–‡Ž›—•‡†–‘’—”‹ˆ›•‘Ž—„Ž‡’”‘–‡‹•Ǥ
Solubilizationofproteinsfrominclusionbodies
E. coli ͳͲȀ’ǦȽ” …‡ŽŽ• ™‡”‡ †‹•”—’–‡† ƒ• †‡•…”‹„‡† ˆ‘” –Š‡ ‡š–”ƒ…–‹‘ ‘ˆ •‘Ž—„Ž‡
’”‘–‡‹•Ǥ ˆ–‡” •‘‹…ƒ–‹‘ǡ –Š‡ ‡š–”ƒ…– ™ƒ• …‡–”‹ˆ—‰‡† ƒ– ͶǡͲͲͲ š‰ ȋʹͲ ‹ǡ Ͷ ιȌǡ ƒ† –Š‡
’‡ŽŽ‡–ȋ…‘–ƒ‹‹‰–Š‡‹…Ž—•‹‘„‘†‹‡•Ȍ™ƒ•™ƒ•Š‡†–™‹…‡™‹–ŠʹͲͲ”‹•ǦŽȋ’ͺȌǡͷͲͲ
 ƒŽǡ ͳ  ǡ ͲǤͳ Ψ ȋ˜Ȁ˜Ȍ ”‹–‘ǦǦͳͲͲǤ ˆ–‡” –Š‡ •‡…‘† ™ƒ•Šǡ –Š‡ ’‡ŽŽ‡– ™ƒ•
͹
Šƒ”˜‡•–‡† ƒ† •–‘”‡† ƒ– ȂʹͲ ι —–‹Ž —•‡†Ǥ ‘ •‘Ž—„‹Ž‹œ‡ ’”‘–‡‹•ǡ ‹…Ž—•‹‘ „‘†‹‡• ™‡”‡
”‡•—•’‡†‡†‹ʹͲͲ”‹•ǦŽȋ’ͺȌǡͷͲͲƒŽǡͷȾǦ‡”…ƒ’–‘‡–Šƒ‘Žǡ͸—”‡ƒǡ
—•‹‰ͳͲͲ‘ˆ„—ˆˆ‡”’‡”‰ȋ™‡–™‡‹‰–ŠȌ‘ˆ•ƒ’Ž‡Ǥˆ–‡”‹…—„ƒ–‹‘ƒ–”‘‘–‡’‡”ƒ–—”‡
ˆ‘”͵Š—†‡”ƒ‰‡–‹…•–‹””‹‰ǡ–Š‡•‘Ž—–‹‘™ƒ•…‡–”‹ˆ—‰‡†ƒ–ͳ͸ǡͲͲͲš‰ȋʹͲ‹ǡʹͲιȌǡƒ†
–Š‡ ’‡ŽŽ‡– ™ƒ• †‹•…ƒ”†‡†ǡ ›‹‡Ž†‹‰ ƒ •—’‡”ƒ–ƒ– …‘–ƒ‹‹‰ ͷͲ ‰ ‘ˆ ’”‘–‡‹• ’‡” ‰ ‘ˆ
‹…Ž—•‹‘ „‘†‹‡•Ǥ ‘ ”‡ˆ‘Ž† ’”‘–‡‹•ǡ –Š‡ •—’‡”ƒ–ƒ– ™ƒ• •—„Œ‡…–‡† –‘ ˆ‘—” †‹ƒŽ›•‹• •–‡’•ǡ
ƒ‰ƒ‹•–ǣ‹ȌʹͲͲ”‹•ǦŽȋ’ͺȌǡͷͲͲƒŽǡͷȾǦ‡”…ƒ’–‘‡–Šƒ‘Žǡʹ‰ŽʹǡͶ
—”‡ƒǢ‹‹ȌͳͲͲ”‹•ǦŽǡͷͲͲƒŽǡͷȾǦ‡”…ƒ’–‘‡–Šƒ‘Žǡʹ‰Žʹǡʹ—”‡ƒǢ‹‹‹Ȍ
ͷͲ”‹•ǦŽǡʹͲͲƒŽǡͷȾǦ‡”…ƒ’–‘‡–Šƒ‘Žǡʹ‰Žʹǡͳ—”‡ƒǢ‹˜Ȍƒ†ͷͲ
”‹•ǦŽǡͷͲƒŽǡͷȾǦ‡”…ƒ’–‘‡–Šƒ‘Žǡʹ‰ŽʹǡͳͲΨȋ˜Ȁ˜Ȍ‰Ž›…‡”‘ŽǤŠ‡
†‹ƒŽ›•‹•–—„‡…‘–ƒ‹‡†͵Ͳ‘ˆ•ƒ’Ž‡’‡”Ž‹–‡”‘ˆ‡š–‡”ƒŽ„—ˆˆ‡”Ǥ
Purificationofsolubleproteins
Š‡Ƚ•—„—‹–‘ˆE.coli‘Ž™ƒ•’—”‹ˆ‹‡†ƒ•’”‡˜‹‘—•Ž›†‡•…”‹„‡†ȏͶ͹Ȑǡƒ†–Š‡•ƒ‡
’”‘…‡†—”‡ ™ƒ• —•‡† –‘ ‹•‘Žƒ–‡ Š‘‘‰‡‡‘—• Ƚ”Ǥ ”‹‡ˆŽ›ǡ –Š‡ •‘Ž—„Ž‡ ’”‘–‡‹ ‡š–”ƒ…–• ™‡”‡
•—„Œ‡…–‡†–‘ƒ‹‘Ǧ‡š…Šƒ‰‡…Š”‘ƒ–‘‰”ƒ’Š›ȋǦ‡’Šƒ”‘•‡ǡͷͲ…‘Ž—Ȍ‹—ˆˆ‡”ǡ
ƒ’’Ž›‹‰ ƒ Ž‹‡ƒ” ͷͲǦ͸ͲͲ  ƒŽ ‰”ƒ†‹‡–Ǥ Š‡ „‡•– ˆ”ƒ…–‹‘•ǡ ƒ……‘”†‹‰ –‘ Ǧ
ƒƒŽ›•‹•ǡ™‡”‡’‘‘Ž‡†ǡ†‡•ƒŽ–‡†ǡ…‘…‡–”ƒ–‡†ǡ•—’’Ž‡‡–‡†™‹–ŠʹͲΨȋ˜Ȁ˜Ȍ‰Ž›…‡”‘Žǡͷ
‰Žʹǡ ƒ† Ž‘ƒ†‡† ‘–‘ ƒ ‹„ƒ…”‘ Ž—‡ …‘Ž— ȋͳͷ ȌǤ ˆ–‡” ‡š–‡•‹˜‡ ™ƒ•Š‹‰ ‘ˆ –Š‡
…‘Ž— ™‹–Š —ˆˆ‡” ȋ—ˆˆ‡” …‘–ƒ‹‹‰ ʹͲ Ψ ‰Ž›…‡”‘Ž ƒ† ͷ  ‰ŽʹȌǡȽ” ™ƒ•‡Ž—–‡†
™‹–Š –Š‡ •ƒ‡ „—ˆˆ‡” …‘–ƒ‹‹‰ ͳ ƒŽǤ Š‡ ‡Ž—–‡† ’‡ƒ ™ƒ• †‡•ƒŽ–‡† ƒ† Ž‘ƒ†‡† ‘–‘ ƒ
‹”ƒ’‡’ƒ”‹…‘Ž—ȋͷȌǡ–‘™Š‹…ŠƒŽ‹‡ƒ”ͷͲǦ͸ͲͲ‰”ƒ†‹‡–‘ˆƒŽ™ƒ•ƒ’’Ž‹‡†Ǥ
Š‡„‡•–ˆ”ƒ…–‹‘•™‡”‡”‡…‘†‹–‹‘‡†–‘ͷͲƒŽ™‹–Š—ˆˆ‡”…‘–ƒ‹‹‰ʹͲΨ‰Ž›…‡”‘Žǡ
ƒ†–Š‡›™‡”‡ˆ‹ƒŽŽ›•—„Œ‡…–‡†–‘ƒ•‡…‘†ƒ‹‘‡š…Šƒ‰‡…”Š‘ƒ–‘‰”ƒ’Š›ǡ—•‹‰ƒ‹”ƒ’
…‘Ž—ȋͷȌǤŠ‡„‡•–ˆ”ƒ…–‹‘•ǡ…‘–ƒ‹‹‰’ƒ”–‹ƒŽŽ›’—”‹ˆ‹‡†Ƚ”ǡ™‡”‡’‘‘Ž‡†ƒ†•–‘”‡†
ƒ–ȂʹͲ闐–‹Ž—•‡†Ǥ
ͺ
PurificationofȽDrfrominclusionbodies
Š‡”‡ˆ‘Ž†‡†•ƒ’Ž‡™ƒ•Ž‘ƒ†‡†‘–‘ƒ‹„ƒ…”‘Ž—‡…‘Ž—ȋͳͷȌǡƒ†ƒˆ–‡”ƒ™ƒ•Š‹‰
•–‡’ ȋ͸ …‘Ž— ˜‘Ž—‡•Ȍǡ Ƚ” ™ƒ• ‡Ž—–‡† ™‹–Š —ˆˆ‡” …‘–ƒ‹‹‰ ͳ ƒŽǤ ˆ–‡” †‹ƒŽ›•‹•
ƒ‰ƒ‹•– —ˆˆ‡” …‘–ƒ‹‹‰ ʹͲ Ψ ‰Ž›…‡”‘Žǡ –Š‡ •ƒ’Ž‡ ™ƒ• Ž‘ƒ†‡† ‘–‘ ƒ ‹”ƒ’ ‡’ƒ”‹
…‘Ž—ȋͷȌƒ†•—„Œ‡…–‡†–‘ƒŽ‹‡ƒ”ͷͲǦ͸ͲͲƒŽ‰”ƒ†‹‡–ǤŠ‡„‡•–ˆ”ƒ…–‹‘•™‡”‡
’‘‘Ž‡†ƒ†•–‘”‡†ƒ–ȂʹͲ闐–‹Ž—•‡†Ǥ
MassSpectrometry
Ž—–‹‘‘ˆ’”‘–‡‹•ˆ”‘ƒ…”›Žƒ‹†‡‰‡Ž•ǡ–”›’•‹†‹‰‡•–‹‘ǡƒ†•‡’ƒ”ƒ–‹‘‘ˆ’‡’–‹†‡•™‡”‡
’‡”ˆ‘”‡†ƒ•’”‡˜‹‘—•Ž›†‡•…”‹„‡†ȏͳ͹ȐǤŠ‡”‡•—Ž–‹‰’‡’–‹†‡•™‡”‡ƒƒŽ›œ‡†„›ǦȀ
—•‹‰ƒ”„‹–”ƒ’‹•–”—‡–ȋŠ‡”‘‹•Š‡”ǡƒŽ–ŠƒǡǡȌ‡“—‹’‡‡†™‹–Šƒƒ‘Ǧ
•‘—”…‡ …‘—’Ž‡† ™‹–Š ƒ ƒ‘Ǧ…“—‹–› …ƒ’‹ŽŽƒ”› ȋƒ–‡”•ǡ ‹Žˆ‘”†ǡ ǡ ȌǤ ”‹‡ˆŽ›ǡ
’‡’–‹†‡•™‡”‡•‡’ƒ”ƒ–‡†™‹–Šƒ…ƒ’‹ŽŽƒ”›ͳͺ…‘Ž—ȋͲǤͲ͹ͷšͳͲͲǡͳǤ͹Ɋǡƒ–‡”•Ȍ
—•‹‰ƒ“—‡‘—•ͲǤͳ؈‘”‹…ƒ…‹†ȋȌƒ†͵…‘–ƒ‹‹‰ͲǤͳ؈‘”‹…ƒ…‹†ȋȌƒ•‘„‹Ž‡
’Šƒ•‡•Ǥ‡’–‹†‡•™‡”‡‡Ž—–‡†„›‡ƒ•‘ˆƒŽ‹‡ƒ”‰”ƒ†‹‡–ˆ”‘ͷ–‘ͷͲΨ‘ˆ‹ͻͲ‹ǡƒ–ƒ
͵ͲͲȀ‹ˆŽ‘™”ƒ–‡Ǥƒ•••’‡…–”ƒ™‡”‡ƒ…“—‹”‡†‘˜‡”ƒm/z”ƒ‰‡ˆ”‘ͶͲͲ–‘ͳͺͲͲǤ‘
ƒ…Š‹‡˜‡’”‘–‡‹‹†‡–‹ˆ‹…ƒ–‹‘ǡƒ†Ȁ†ƒ–ƒ—†‡”™‡–ƒ•…‘–‡ƒ”…Š‰‹‡•‘ˆ–™ƒ”‡
ƒƒŽ›•‹• –‘ ‹–‡””‘‰ƒ–‡ –Š‡ ƒ–‹‘ƒŽ ‡–‡” ˆ‘” ‹‘–‡…Š‘Ž‘‰› ˆ‘”ƒ–‹‘ ‘”‡†—†ƒ–
ȋ”Ȍ’”‘–‡‹†ƒ–ƒ„ƒ•‡Ǥƒ”ƒ‡–‡”••‡–•™‡”‡ǣ–”›’•‹…Ž‡ƒ˜ƒ‰‡Ǣ…ƒ”„ƒ‹†‘‡–Š›Žƒ–‹‘‘ˆ
…›•–‡‹‡• ƒ• ƒ ˆ‹š‡† ‘†‹ˆ‹…ƒ–‹‘ ƒ† ‡–Š‹‘‹‡ ‘š‹†ƒ–‹‘ ƒ• ƒ ˜ƒ”‹ƒ„Ž‡ ‘†‹ˆ‹…ƒ–‹‘Ǣ ƒ
ƒš‹— ‘ˆ –™‘ ‹••‡† …Ž‡ƒ˜ƒ‰‡•Ǣ ˆƒŽ•‡ †‹•…‘˜‡”› ”ƒ–‡ǡ …ƒŽ…—Žƒ–‡† „› •‡ƒ”…Š‹‰ –Š‡ †‡…‘›
†ƒ–ƒ„ƒ•‡ǡͲǤͲͷǤ
Enzymeassays
›’‡ƒ…–‹˜ƒ–‡†…ƒŽˆǦ–Š›—•™ƒ•‘„–ƒ‹‡†ˆ”‘‹‰ƒǦŽ†”‹…Šȋ–Ǥ‘—‹•ǡǡȌǤE.
coli‡…”‡…‘„‹ƒ•‡ƒ††•‹š–—”‡ȋ†ǡ†
ǡ†ǡƒ††ǡͳͲ‡ƒ…ŠȌ™‡”‡
’—”…Šƒ•‡†ˆ”‘ȋ’•™‹…ŠǡǡȌǤŽ‹‰‘—…Ž‡‘–‹†‡•ǡ–‘„‡ƒ‡ƒŽ‡†ƒ†—•‡†ƒ•
ͻ
•—„•–”ƒ–‡•ǡ ™‡”‡ •›–Š‡•‹œ‡† „› ‡…”‹’– ȋ‹•…ƒ–ƒ™ƒ›ǡ ǡ ȌǤ ‘Ž›‡”ƒ•‡ ƒ…–‹˜‹–›
™ƒ• †‡–‡”‹‡† ƒ……‘”†‹‰ –‘ ‘—” ’”‡˜‹‘•—Ž› †‡•…”‹„‡† ‡œ›‡Ǧ…‘—’Ž‡† ƒ••ƒ›ǡ ™Š‹…Š ™ƒ•
†‡•‹‰‡†ˆ‘”–Š‡…‘–‹—‘—••’‡…–”‘’Š‘–‘‡–”‹…†‡–‡…–‹‘‘ˆ’›”‘’Š‘•’Šƒ–‡Ȁ’Š‘•’Šƒ–‡ȏͶͺȐǤ
”‹‡ˆŽ›ǡ”‡ƒ…–‹‘•™‡”‡ƒ••ƒ›‡†‹ͳͲͲ”‹•ǦŽȋ’ͺȌǡͷ‰ŽʹǡͲǤʹͷ‹‘•‹‡ǡͳͲͲ
Ɋ †ȋ•Ȍǡ ƒ† ͳͲǡ ͷͲǡ ƒ† ͷͲͲ Ȁ ‘ˆ ‹‘”‰ƒ‹… ’›”‘’Š‘•’Šƒ–ƒ•‡ ȋƒ•‡Ȍǡ ’—”‹‡
—…Ž‡‘•‹†‡’Š‘•’Š‘”›Žƒ•‡ȋƒ•‡Ȍǡƒ†ƒ–Š‹‡š‹†ƒ•‡ȋȌǡ”‡•’‡…–‹˜‡Ž›ǤŠ‡—”‹…ƒ…‹†
‰‡‡”ƒ–‡† „› ™ƒ• †‡–‡…–‡† ƒ– ʹͻ͵ ǡ ƒ† ’Š‘•’Šƒ–‡ …‘…‡–”ƒ–‹‘• ™‡”‡ …ƒŽ…—Žƒ–‡†
ƒ••—‹‰ƒ‘Žƒ”‡š–‹…–‹‘…‘‡ˆˆ‹…‹‡–ˆ‘”—”‹…ƒ…‹†‡“—ƒŽ–‘ͳʹǡ͸ͲͲǦͳ…ǦͳȏͶͻȐǤŠ‡ƒ•‡
ƒ…–‹˜‹–›‘ˆ‡…™ƒ•†‡–‡”‹‡††‡–‡…–‹‰„‘–Š–Š‡‘”–Š‘’Š‘•’Šƒ–‡ƒ†–Š‡‰‡‡”ƒ–‡†Ǥ
‘…‘–‹—‘—•Ž›†‡–‡”‹‡–Š‡”‡Ž‡ƒ•‡‘ˆǡ–Š‡”‡ƒ…–‹‘‹š–—”‡…‘–ƒ‹‡†ͳͲͲ”‹•Ǧ
Ž ȋ’ ͺȌǡ ͳͲ  ‰Žʹǡ ʹͲ  Žǡ ͲǤͷ  ’Š‘•’Š‘‡‘Ž’›”—˜ƒ–‡ ȋȌǡ ͳǤͷ Ȁ ‘ˆ
›”—˜ƒ–‡‹ƒ•‡ȋȌǡͲǤʹͷȾǦǡƒ†͸Ȁ‘ˆƒ…–ƒ–‡†‡Š›†”‘‰‡ƒ•‡ȋȌǤ›–Š‹•
‡ƒ•ǡ–Š‡…‘•—‡†„›‡…‹•”‡‰‡‡”ƒ–‡†ˆ”‘ƒ†ǡ™Š‹…Š‹•…‘˜‡”–‡†–‘
’›”—˜ƒ–‡„›Ǣˆ‹ƒŽŽ›ǡ’›”—˜ƒ–‡‹•”‡†—…‡†ǡ‹–Š‡’”‡•‡…‡‘ˆȾǦǡ–‘Žƒ…–ƒ–‡Ǥ‡ƒ…–‹‘•
™‡”‡ ‘‹–‘”‡† ƒ– ͵ͶͲ ǡ ƒ† –Š‡ ‘Žƒ” ‡š–‹…–‹‘ …‘‡ˆˆ‹…‹‡– ‘ˆ ȾǦ ™ƒ• ƒ••—‡†
‡“—ƒŽ–‘͸ǡʹͲͲǦͳ…ǦͳǤ
Š‡ Žƒ‰ –‹‡ „‡ˆ‘”‡ •–‡ƒ†›Ǧ•–ƒ–‡ ‹–‡”‡†‹ƒ–‡ …‘…‡–”ƒ–‹‘• ƒ”‡ ”‡ƒ…Š‡†ǡ ƒ• ™‡ ‘„•‡”˜‡†
Š‡”‡ǡ ‹• ™‡ŽŽ ‘™ ˆ‘” ‡œ›‡ …‘—’Ž‡† ƒ••ƒ›• ȏͷͲȐǤ …–‹˜‹–‹‡• ™‡”‡ †‡–‡”‹‡† „›
…‘•‹†‡”‹‰ –Š‡ ‹–‡”˜ƒŽ –‹‡ǡ •—„•‡“—‡– –‘ –Š‡ Žƒ‰ǡ †—”‹‰ ™Š‹…Š œ‡”‘Ǧ‘”†‡” ‹‡–‹…• ™ƒ•
†‡–‡…–‡†Ǥ
ŽŽ –Š‡ ƒ••ƒ›• ™‡”‡ ’‡”ˆ‘”‡† —•‹‰ ƒ ƒ”› ͵ͲͲ Ǧ •’‡…–”‘’Š‘–‘‡–‡”Ǥ ”‘–‡‹
…‘…‡–”ƒ–‹‘ ™ƒ• †‡–‡”‹‡† ƒ……‘”†‹‰ –‘ ”ƒ†ˆ‘”† ȏͷͳȐǡ —•‹‰ „‘˜‹‡ •‡”— ƒŽ„—‹‡ ƒ•
•–ƒ†ƒ”†Ǥ
ͳͲ
RESULTS
OverexpressionofsolubleD.radioduransDnaEpolymerase
‡ˆ‹”•–ƒ––‡’–‡†–Š‡‘˜‡”‡š’”‡••‹‘‘ˆD.radioduransȽ”‹•‘Ž—„Ž‡ˆ‘”Ǥ‘–Š‹•ƒ‹ǡ™‡
–”ƒ•ˆ‘”‡† E. coli ͳͲ ™‹–Š ’ǦȽ”ǡ ƒ† ™‡ •‡ƒ”…Š‡† ˆ‘” ‰”‘™–Š ƒ† ‹†—…–‹‘
…‘†‹–‹‘• …‘””‡•’‘†‹‰ –‘ ‘˜‡”‡š’”‡••‹‘ ›‹‡Ž†• …‘’ƒ–‹„Ž‡ ™‹–Š –Š‡ ’—”‹ˆ‹…ƒ–‹‘ ‘ˆ –Š‡
–ƒ”‰‡– ’”‘–‡‹Ǥ ‘™‡˜‡”ǡ —•‹‰ E. coli ͳͲȀ’ǦȽ” ™‡ ™‡”‡ ‘– ƒ„Ž‡ –‘ ‘„–ƒ‹
•ƒ–‹•ˆƒ…–‘”› ƒ‘—–• ‘ˆ •‘Ž—„Ž‡ Ƚ” ȋ†ƒ–ƒ ‘– •Š‘™ȌǤ  –Š‡ …‘–”ƒ”›ǡ ™Š‡ E. coli
ʹͷͻͻ͵Ȁ’ǦȽ”™ƒ•‰”‘™ƒ–͵͹郐†‹†—…‡†ˆ‘”͵Šƒ––Š‡•ƒ‡–‡’‡”ƒ–—”‡ǡŠ‹‰Š
‘˜‡”‡š’”‡••‹‘ ‘ˆ •‘Ž—„Ž‡ Ƚ” ™ƒ• ‘„•‡”˜‡† ȋ—’’Ž‡‡–ƒ”› ‹‰—”‡ ͳȌǤ ‘—„Ž‡ ’”‘–‡‹
‡š–”ƒ…–•™‡”‡ƒ……‘”†‹‰Ž›’”‡’ƒ”‡†ˆ”‘–Š‹•„ƒ…–‡”‹ƒŽŠ‘•–ǡƒ†–Š‡’—”‹ˆ‹…ƒ–‹‘‘ˆȽ™ƒ•
’—”•—‡† „› ˆ‘—” …‘•‡…—–‹˜‡ …Š”‘ƒ–‘‰”ƒ’Š‹… •–‡’• ȋ•‡‡ ‡–Š‘†•ȌǤ › –Š‹• ‡ƒ•ǡ ™‡
•‹‰‹ˆ‹…ƒ–Ž› ’—”‹ˆ‹‡† Ƚ” ȋ—’’Ž‡‡–ƒ”› ‹‰—”‡ ͳǡ ƒ„Ž‡ ͳȌǡ „—– ™‡ …‘—Ž† ‘– †‹••‘…‹ƒ–‡
ˆ”‘ –Š‡ ‘Ž›‡”ƒ•‡ ƒ ƒ……‘’ƒ›‹‰ …‘–ƒ‹ƒ–ǡ ™Š‹…Š ™ƒ• ‹†‡–‹ˆ‹‡† „› ƒ••
•’‡…–”‘‡–”› ƒ• E. coli ‘Ž›‡”ƒ•‡ ȋƒ„Ž‡• ʹǦ͵ȌǤ ‘•‹†‡”‹‰ –Šƒ– –Š‡ …‘…‡–”ƒ–‹‘ ‘ˆ
–Š‹•’ƒ”–‹ƒŽŽ›’—”‹ˆ‹‡†Ƚ”…ƒ‘–„‡“—ƒ–‹–ƒ–‡†ǡ™‡—•‡†–Š‹•‡œ›‡’”‡’ƒ”ƒ–‹‘–‘’‡”ˆ‘”
“—ƒŽ‹–ƒ–‹˜‡ƒ••ƒ›•ǡ–Š‡ƒ‹‘ˆ™Š‹…Š™ƒ•–‘…‘ˆ‹”™Šƒ–™‡‘„•‡”˜‡†—•‹‰”‡ˆ‘Ž†‡†Ƚ”ȋ•‡‡
„‡Ž‘™ȌǤ
RefoldingandpurificationofD.radioduransDnaEpolymerasefrominclusionbodies
‘’—”•—‡–Š‡‹•‘Žƒ–‹‘‘ˆŠ‹‰ŠŽ›’—”‹ˆ‹‡†Ƚ”ǡ†‡˜‘‹†‘ˆE.coli‘Ž›‡”ƒ•‡ǡ™‡†‡…‹†‡†–‘
ƒ––‡’––Š‡”‡ˆ‘Ž†‹‰‘ˆ–Š‡‡œ›‡ˆ”‘‹…Ž—•‹‘„‘†‹‡•ǤŠ‡‹†—…–‹‘ˆ‘”ͳͷŠƒ–͵͹ι‘ˆ
E. coli ͳͲȀ’ǦȽ” ›‹‡Ž†‡† ͲǤͻ ‰ ȋ™‡– ™‡‹‰Š–Ȍ ‘ˆ ‹…Ž—•‹‘ „‘†‹‡• ’‡” Ž‹–‡” ‘ˆ …—Ž–—”‡Ǥ
’‘•‘Ž—„‹Ž‹œƒ–‹‘™‹–Š—”‡ƒ͸ǡƒ†ˆ‘—”…‘•‡…—–‹˜‡†‹ƒŽ›•‹••–‡’•’‡”ˆ‘”‡†ƒ•†‡•…”‹„‡†
„› ”‘™ ‡– ƒŽǤ ˆ‘” ›‡ƒ•– ’‘Ž›‡”ƒ•‡ Ɂ ȏͷʹȐǡ ™‡ ˆ‹ƒŽŽ› ‘„–ƒ‹‡† ʹ͹ ‰ ‘ˆ •‘Ž—„Ž‡
’”‘–‡‹• ’‡” ‰ ‘ˆ ‹…Ž—•‹‘ „‘†‹‡•ǡ ƒ‘‰ ™Š‹…Š Ƚ” ™ƒ• Š‹‰ŠŽ› ƒ„—†ƒ–Ǥ Š‹• ”‡ˆ‘Ž†‡†
ͳͳ
•ƒ’Ž‡ ™ƒ• –Š‡ •—„Œ‡…–‡† –‘ ‹„ƒ…”‘ Ž—‡ ƒ† ‹”ƒ’ ‡’ƒ”‹ ƒˆˆ‹‹–› …Š”‘ƒ–‘‰”ƒ’Š›
ȋ—’’Ž‡‡–ƒ”›‹‰—”‡ʹȌǤ‹–Š„‘–Š…Š”‘ƒ–‘‰”ƒ’Š‹…•—’’‘”–•ǡƒ…‘•‹•–‡–ƒ‘—–‘ˆȽ”
™ƒ•Ž‘•–™‹–Š–Š‡ˆŽ‘™Ǧ–Š”‘—‰Šǡ‘•–Ž‹‡Ž›…‘–ƒ‹‹‰‹’”‘’‡”Ž›”‡ˆ‘Ž†‡†‡œ›‡Ǥ”‘ʹ͹
‰‘ˆ†‹ƒŽ›œ‡†’”‘–‡‹•ǡ™‡™‡”‡ƒ„Ž‡–‘”‡…‘˜‡”͵Ǥ͸‰‘ˆ”‡ˆ‘Ž†‡†ƒ†’—”‹ˆ‹‡†Ƚ”Ǥ
DNAPolymeraseactivityofrefoldedȽDrandȽEc
‡…‘’ƒ”‡†–Š‡…ƒ–ƒŽ›–‹…’‡”ˆ‘”ƒ…‡•‘ˆ”‡ˆ‘Ž†‡†Ƚ”™‹–Š–Š‘•‡‘ˆ–Š‡™‡ŽŽǦ‘™Ƚ…Ǥ
‘–Š‹•ƒ‹ǡ™‡ˆ‹”•–ƒ••ƒ›‡†–Š‡’‘Ž›‡”ƒ•‡ƒ…–‹˜‹–›‘ˆ„‘–Š‡œ›‡•ƒ––Š‡‡š’‡•‡‘ˆƒ
ͶͲ‡”–‡’Žƒ–‡ƒ‡ƒŽ‡†–‘ƒͳͷ‡”’”‹‡”ǡ–Š‡‡š–‡•‹‘‘ˆ™Š‹…Š‡š…Ž—•‹˜‡Ž›†‡’‡†•
‘ † ȋ‹‰—”‡ ͳȌǤ Š‡ ‡š–‡•‹‘ ‘ˆ ™ƒ• ‘‹–‘”‡† „› ‘—” ’”‡˜‹‘—•Ž› †‡•…”‹„‡†
‡œ›‡Ǧ…‘—’Ž‡† ƒ••ƒ› ȋ ƒ••ƒ›ǡ ȏͶͺȐȌǡ ™Š‹…Š ”‡Ž‹‡• ‘ ȋ…Š‡‡ ͳȌǣ ‹Ȍ ‹‘”‰ƒ‹…
’›”‘’Š‘•’Šƒ–ƒ•‡ȋƒ•‡Ȍǡ–Š‡ƒ…–‹‘‘ˆ™Š‹…Š”‡Ž‡ƒ•‡•‘”–Š‘’Š‘•’Šƒ–‡ƒ––Š‡‡š’‡•‡‘ˆ–Š‡
’›”‘’Š‘•’Šƒ–‡‰‡‡”ƒ–‡†„›ƒ‘ŽǢ‹‹Ȍ’—”‹‡—…Ž‡‘–‹†‡’Š‘•’Š‘”›Žƒ•‡ȋƒ•‡Ȍǡ™Š‹…Š
‹ –Š‡ ’”‡•‡…‡ ‘ˆ ‹‘•‹‡ ƒ† ‘”–Š‘’Š‘•’Šƒ–‡ǡ …ƒ–ƒŽ›œ‡• –Š‡ ”‡Ž‡ƒ•‡ ‘ˆ ”‹„‘•‡ǦͳǦ’Š‘•’Šƒ–‡
ƒ† Š›’‘šƒ–Š‹‡Ǣ ‹‹‹Ȍ ƒ† šƒ–Š‹‡ ‘š‹†ƒ•‡ ȋȌǡ ˆ‹ƒŽŽ› …‘˜‡”–‹‰ Š›’‘šƒ–Š‹‡ –‘ —”‹…
ƒ…‹†ǡ™Š‹…Š‹•…‘˜‡‹‡–Ž›†‡–‡…–ƒ„Ž‡ƒ–ʹͻ͵ȏͶͻȐǤ†‡”–Š‡•‡…‘†‹–‹‘•ǡ–Š‡ƒ…–‹˜‹–›‘ˆ
Ƚ” ™ƒ• ˆ‘—† –‘ „‡ ƒ„‘—– ͻͲ –‹‡• Ž‘™‡” ™Š‡ …‘’ƒ”‡† ™‹–Š –Šƒ– ‘ˆ –Š‡ E. coli ‡œ›‡
ȋͲǤͳͻƒ†ͳ͹Ȁ•ǡ”‡•’‡…–‹˜‡Ž›ǡ‹‰—”‡•ʹǦʹǡƒ†—’’Ž‡‡–ƒ”›‹‰—”‡•͵Ǧ͵ȌǤ‘™‡˜‡”ǡ
‹–™ƒ•ƒŽ•‘‘„•‡”˜‡†–Šƒ–Ƚ”‹••‹‰‹ˆ‹…ƒ–Ž›•–‹—Žƒ–‡†„›‹‘‹…•–”‡‰–Šǡ‹–•’‘Ž›‡”ƒ•‡
ƒ…–‹˜‹–› „‡‹‰ …ƒǤ ͶǦˆ‘Ž† Š‹‰Š‡” ‹ –Š‡ ’”‡•‡…‡ ‘ˆ ʹͲͲ  ƒŽ ‘” Ž ȋ‹‰—”‡ ʹǡ ƒ†
—’’Ž‡‡–ƒ”› ‹‰—”‡ ͵ȌǤ  –Š‡ …‘–”ƒ”›ǡ Ƚ… ™ƒ• †”ƒƒ–‹…ƒŽŽ› ‹Š‹„‹–‡†ǡ ‹Ǥ‡Ǥ ͵ͲǦˆ‘Ž†ǡ „›
ʹͲͲ  Ž ȋ‹‰—”‡ ʹǡ ƒ† —’’Ž‡‡–ƒ”› ‹‰—”‡ ͵ȌǤ Š‡”‡ˆ‘”‡ǡ —†‡” –Š‡•‡ …‘†‹–‹‘•
Ƚ”’‡”ˆ‘”••Ž‹‰Š–Ž›„‡––‡”–Šƒ‹–•E.coliŠ‘‘Ž‘‰—‡Ǥ‘”‡‘˜‡”ǡ™Š‡…ƒŽˆǦ–Š›—•ƒ…–‹˜ƒ–‡†
™ƒ• —•‡† ƒ• •—„•–”ƒ–‡ǡ ƒ† ƒŽŽ –Š‡ ˆ‘—” †• ™‡”‡ ’”‡•‡– ‹ ”‡ƒ…–‹‘ ‹š–—”‡•ǡ
…‘’ƒ”ƒ„Ž‡ ƒ…–‹˜‹–‹‡• ™‡”‡ ‘„•‡”˜‡† ‹ –Š‡ ’”‡•‡…‡ ‘ˆ Ƚ” ‘” Ƚ… ȋͲǤʹͷ ƒ† ͲǤ͵ Ȁ•ǡ
”‡•’‡…–‹˜‡Ž›ǡ ‹‰—”‡ ʹǡ ƒ† —’’Ž‡‡–ƒ”› ‹‰—”‡ ͵ȌǤ – ™ƒ• ’”‡˜‹‘•—Ž› ”‡’‘”–‡† –Šƒ– D.
ͳʹ
radiodurans‘Ž•ƒ†ƒ”‡’‘•‹–‹˜‡Ž›ƒˆˆ‡…–‡†„›ʹΪȏͶʹǡͶͶȐǤ……‘”†‹‰Ž›ǡ™‡ƒ••ƒ›‡†
Ƚ” ‹ –Š‡ ’”‡•‡…‡ ‘ˆ ƒ‰ƒ‡•‡ǡ –Š‡ ͶͲ‡” †•ǡ ƒ† †Ǥ —”’”‹•‹‰Ž›ǡ ƒ •–”‘‰
ƒ…–‹˜ƒ–‹‘‘ˆȽ”™ƒ•‘„•‡”˜‡†ǡ›‹‡Ž†‹‰ƒƒ…–‹˜‹–›‡“—ƒŽ–‘ͷǤͷȀ•ȋ—’’Ž‡‡–ƒ”›‹‰—”‡
͵Ȍǡ ™Š‹Ž‡ ƒ •ƒŽŽ ‡ˆˆ‡…–ǡ ‹ˆ ƒ›ǡ ™ƒ• †‡–‡…–‡† ‹ –Š‡ ’”‡•‡…‡ ‘ˆ Ƚ… —†‡” –Š‡ •ƒ‡
…‘†‹–‹‘•ȋ†ƒ–ƒ‘–•Š‘™ȌǤ
AcontinuousassayforrecombinationǦdependentDNApolymeraseactivity
‡ –Š‘—‰Š– ‹– ‘ˆ ‹–‡”‡•– –‘ ƒ••ƒ› –Š‡ ƒ…–‹˜‹–› ‘ˆ Ƚ” ƒ† Ƚ… —†‡” …‘†‹–‹‘• ”‡“—‹”‹‰
”‡…‘„‹ƒ–‹‘ –‘ ‰‡‡”ƒ–‡ ƒ †• •—„•–”ƒ–‡Ǥ ……‘”†‹‰Ž›ǡ ™‡ †‡•‹‰‡† –Š”‡‡
‘Ž‹‰‘†‡‘š›—…Ž‡‘–‹†‡•ǡ–™‘‘ˆ™Š‹…Š–‘„‡ƒ‡ƒŽ‡†‹–‘ƒŽ‹‡ƒ”Ͷͺ‡”†•ǡƒ†–Š‡–Š‹”†
–‘„‡—•‡†ƒ•͸Ͳ‡”••‹–Š‡ƒ••ƒ›ȋ‹‰—”‡ͳȌǤ›–Š‹•‡ƒ•ǡ’‘Ž›‡”ƒ•‡ƒ…–‹˜‹–›
•Š‘—Ž† ‘– „‡ ‘„•‡”˜‡† ƒ– –Š‡ ‡š’‡•‡ ‘ˆ †ǡ —Ž‡•• ƒ ’”‡˜‹‘—• ”‡…‘„‹ƒ–‹‘ •–‡’
„‡–™‡‡–Š‡Ͷͺ‡”†•ƒ†–Š‡͸Ͳ‡”••‘……—”•ǡ‰‡‡”ƒ–‹‰ƒ’”‹‡”…‘–‹‰—‘—•–‘ƒ
’‘Ž›–‡’Žƒ–‡ȋ‹‰—”‡ͳȌǤ•ƒˆ‹”•––‡•–ǡ™‡ƒ••ƒ›‡†–Š‡„‹†‹‰‡ˆˆ‹…‹‡…›‘ˆE.coli‡…–‘
–Š‡•–‘„‡”‡…‘„‹‡†Ǥ‘–Š‹•ƒ‹ǡ™‡–‘‘ƒ†˜ƒ–ƒ‰‡‘ˆ‘—”ƒ••ƒ›ǡ—•‡ˆ—Ž–‘†‡–‡…–
–Š‡ ‡… ƒ•‡ ƒ…–‹˜‹–› –”‹‰‰‡”‡† „› –Š‡ ƒ••‘…‹ƒ–‹‘ ‘ˆ –Š‹• ‡œ›‡ –‘ Ǥ  ƒ‰”‡‡‡–
™‹–Š ’”‡˜‹‘—• ‘„•‡”˜ƒ–‹‘•ǡ ™‡ †‡–‡…–‡† Š›†”‘Ž›•‹• …‘—’Ž‡† –‘ –Š‡ „‹†‹‰ ‘ˆ ‡… –‘
••ǡ™Š‹Ž‡†•™ƒ•ˆ‘—†–‘„‡ƒ’‘‘”•—„•–”ƒ–‡ȋ‹‰—”‡͵ȌǤ‡–Š‡ƒ••ƒ›‡†–Š‡‡…Ǧ
†‡’‡†‡…‡ ‘ˆ Ƚ… ’‘Ž›‡”ƒ•‡ ƒ…–‹˜‹–›ǡ —•‹‰ „‘–Š Ͷͺ‡” †• ƒ† ͸Ͳ‡” ••ǡ ‹ –Š‡
’”‡•‡…‡ ‘ˆ †‹ˆˆ‡”‡– †•Ǥ • •Š‘™ ‹ ‹‰—”‡• ͵ ƒ† ͵ǡ ‘Ž› ‹ –Š‡ ’”‡•‡…‡ ‘ˆ †
†‘‡•Ƚ…ˆ‡ƒ–—”‡‡…Ǧ†‡’‡†‡–’‘Ž›‡”ƒ•‡ƒ…–‹˜‹–›ǡ™Š‹Ž‡–Š‡ƒ††‹–‹‘‘ˆ†
–‘”‡ƒ…–‹‘
‹š–—”‡• –”‹‰‰‡”• –Š‡ ‡š–‡•‹‘ ‘ˆ ‹†‡’‡†‡–Ž› ‘ˆ –Š‡ ”‡…‘„‹ƒ•‡Ǥ Š‡”‡ˆ‘”‡ǡ –‘
†‡–‡”‹‡ ”‡…‘„‹ƒ–‹‘Ǧ†‡’‡†‡– ’‘Ž›‡”ƒ•‡ ƒ…–‹˜‹–›ǡ ™‡ †‡…‹†‡† –‘ —•‡ ”‡ƒ…–‹‘
‹š–—”‡•…‘–ƒ‹‹‰†•ǡ••ǡ†ǡ‡…ǡƒ†Ǥƒ††‹–‹‘ǡ™‡†‡•‹‰‡†ƒƒ••ƒ›
ˆ‘” –Š‡ •‹—Ž–ƒ‡‘—• †‡–‡…–‹‘ ‘ˆ –Š‡ ƒ•‡ ƒ…–‹˜‹–› ‘ˆ ‡… ƒ† –Š‡ ’‘Ž›‡”ƒ•‡
ƒ…–‹˜‹–›‘ˆȽ…‘”Ƚ”Ǥ—”ƒ••ƒ›†‘‡•‘–‹†‡‡††‹•…”‹‹ƒ–‡„‡–™‡‡‘Žƒ…–‹˜‹–›
ͳ͵
ƒ†–Š‡ƒ•‡ƒ…–‹˜‹–›‘ˆ‡…Ǥ‘™‡˜‡”ǡ™‡–Š‘—‰Š––Šƒ–ƒƒ††‹–‹‘ƒŽ”‡ƒ…–‹‘’ƒ–Š…‘—Ž†
„‡ …‘—’Ž‡† –‘ ‡…ǡ ‹Ǥ‡Ǥ –Š‡ ”‡‰‡‡”ƒ–‹‘ ‘ˆ …ƒ–ƒŽ›œ‡† „› ’›”—˜ƒ–‡ ‹ƒ•‡ ȋȌ ƒ– –Š‡
‡š’‡•‡ ‘ˆ ’Š‘•’‘‡‘Ž’›”—˜ƒ–‡ ȋȌǡ ƒ† –Š‡ ”‡†—…–‹‘ ‘ˆ ’›”—˜ƒ–‡ –‘ Žƒ…–ƒ–‡ „› Žƒ…–ƒ–‡
†‡Š›†”‘‰‡ƒ•‡ȋȌǡ‹–Š‡’”‡•‡…‡‘ˆȾǦȋ…Š‡‡ͳȌǤ•‹‰–Š‹••–”ƒ–‡‰›ǡ–Š‡ƒ…–‹˜‹–›
‘ˆ‡……‘—Ž†„‡‘‹–‘”‡†„›“—ƒ–‹–ƒ–‹‰–Š‡…‘•—’–‹‘‘ˆȾǦǡƒ†‘Žƒ…–‹˜‹–›
™‘—Ž† „‡ ‹†‡–‹ˆ‹‡† ‡•–‹ƒ–‹‰ –Š‡ ƒ‘—– ‘ˆ ‘”–Š‘’Š‘•’Šƒ–‡ Ž‹‡† –‘ ’›”‘’Š‘•’Šƒ–‡
Š›†”‘Ž›•‹•„›ƒ•‡Ǥ–•Š‘—Ž†ƒŽ•‘„‡‡–‹‘‡†–Šƒ––Š‡–™‘‘Žƒ”‡š–‹…–‹‘…‘‡ˆˆ‹…‹‡–•‘ˆ
ȾǦ ƒ† —”‹… ƒ…‹† ƒ”‡ ͸ǡʹͲͲ ƒ† ͳʹǡ͸ͲͲ Ǧͳ…Ǧͳǡ ”‡•’‡…–‹˜‡Ž›Ǥ • •Š‘™ ‹ ‹‰—”‡ Ͷǡ
™Š‡ –Š‡ ƒ„•‘”’–‹‘ •’‡…–”— ‘ˆ ƒ •‘Ž—–‹‘ …‘–ƒ‹‹‰ ••ǡ ‡…ǡ ǡ ‹‘•‹‡ǡ ƒ•‡ǡ
ǡǡȾǦǡǡƒ†™ƒ•”‡…‘”†‡†ƒ•ƒˆ—…–‹‘‘ˆ–‹‡ǡ–Š‡„ƒ†‘ˆ—”‹…ƒ…‹†ƒ–
ʹͻ͵  †‹† ’”‘‰”‡••‹˜‡Ž› ‹…”‡ƒ•‡ǡ ƒ† –Š‡ „ƒ† ‘ˆ ȾǦ †‹† …‘…‘‹–ƒ–Ž› †‡…”‡ƒ•‡ǡ
‹†‹…ƒ–‹‰ –Š‡ ”‡Ž‡ƒ•‡ „› ‡… ‘ˆ ‘”–Š‘Š’‘•’Šƒ–‡ ˆ”‘ Ǥ ‘”‡‘˜‡”ǡ ‹ˆ –Š‡ „•‘”„ƒ…‡
˜ƒŽ—‡•ƒ–ʹͻ͵ƒ†ƒ–͵ͶͲƒ”‡„‘–Š…‘•‹†‡”‡†ƒ•ƒˆ—…–‹‘‘ˆ–‹‡ǡ–Š‡‹‡–‹…•‘ˆ‡…
ƒ…–‹˜‹–›…ƒ„‡”‡Ž‹ƒ„Ž›“—ƒ–‹–ƒ–‡†ȋ‹‰—”‡ͶȌǤ•ƒˆ—”–Š‡”–‡•–‘ˆ‘—”…‘–‹—‘—•ƒ••ƒ›ˆ‘”
”‡…‘„‹ƒ–‹‘Ǧ†‡’‡†‡– ’‘Ž›‡”ƒ•‡ ƒ…–‹˜‹–›ǡ †‡‘‹ƒ–‡† Š‡”‡ ƒ• Ǧ ƒ••ƒ›ǡ ™‡
”‡…‘”†‡†„•‘”’–‹‘•’‡…–”ƒ‘ˆ•‘Ž—–‹‘•…‘–ƒ‹‹‰ƒ…‘’Ž‡–‡”‡ƒ…–‹‘‹š–—”‡ǡ‹Ǥ‡Ǥ†•ǡ
••ǡ†ǡȽǡ‹‘•‹‡ǡƒ•‡ǡƒ•‡ǡǡ‡…ǡǡǡȾǦǡǡƒ†Ǥ†‡”
–Š‡•‡…‘†‹–‹‘•ǡ–Š‡‹…”‡ƒ•‡‹„•‘”’–‹‘ƒ–ʹͻ͵™ƒ•‘”‡–Šƒ–™‹…‡–Š‡„•‘”„ƒ…‡
†‡…”‡ƒ•‡ ƒ– ͵ͶͲ ǡ ‹†‹…ƒ–‹‰ –Š‡ ’”‡•‡…‡ ‘ˆ ‘Ž ƒ…–‹˜‹–› –”‹‰‰‡”‡† „› –Š‡
”‡…‘„‹ƒ–‹‘ ‘ˆ †• ƒ† •• ȋ‹‰—”‡ ͶȌǤ ‘”‡‘˜‡”ǡ ™Š‡ –Š‡ ˆ‹”•– ‹—–‡• ‘ˆ –Š‡
”‡ƒ…–‹‘ ƒ”‡ ƒƒŽ›œ‡†‹ †‡–ƒ‹Žǡ ‹–…ƒ „‡ ‘„•‡”˜‡† –Šƒ– ”‡…‘„‹ƒ–‹‘ ‘……—”• ˆ‹”•–ǡ ƒ† –Š‡
‘Žƒ…–‹˜‹–›–ƒ‡•’Žƒ…‡ȋ‹‰—”‡ͶȌǤ
RecombinationǦdependentpolymeraseactivityofȽEcandȽDr
Š‡”‡…‘„‹ƒ–‹‘Ǧ†‡’‡†‡–’‘Ž›‡”ƒ•‡ƒ…–‹˜‹–›‘ˆȽ”™ƒ•–‡•–‡†™‹–Š–Š‡Ǧƒ••ƒ›ǡ
„›”‡…‘”†‹‰„•‘”’–‹‘•’‡…–”ƒ‡˜‡”›ͳͲ‹ƒˆ–‡”–Š‡”‡ƒ…–‹‘™ƒ••–ƒ”–‡†„›–Š‡ƒ††‹–‹‘
ͳͶ
‘ˆ ‡… ƒ† Ƚ”Ǥ • •Š‘™ ‹ ‹‰—”‡ ͷǡ Ƚ” †‹† ‡ˆˆ‹…‹‡Ž› …ƒ–ƒŽ›œ‡ –Š‡ ‡š–‡•‹‘ ‘ˆ –Š‡
†•‰‡‡”ƒ–‡†„›E.coli‡…Ǥ”‘‹‰—”‡ͷ‹–‹•‹†‡‡†…Ž‡ƒ”Ž›˜‹•‹„Ž‡–Šƒ––Š‡ƒ’Ž‹–—†‡
‘ˆ –Š‡ —”‹… ƒ…‹† „ƒ† †‘‡• ’”‘’–Ž› ‡š…‡‡† ‘”‡ –Šƒ –™‹…‡ –Š‡ ƒ’Ž‹–—†‡ ‘ˆ –Š‡ ‡‰ƒ–‹˜‡
„ƒ†”‡Žƒ–‡†–‘ȾǦ…‘•—’–‹‘ǤŠ‡…‘””‡•’‘†‹‰ȟ„•‘”„ƒ…‡˜ƒŽ—‡•ƒ–ʹͻ͵ƒ†͵ͶͲ
ǡ ƒ• ƒ ˆ—…–‹‘ ‘ˆ –‹‡ǡ ™‡”‡ —•‡† –‘ …ƒŽ…—Žƒ–‡ –Š‡ …‘…‡–”ƒ–‹‘ ‘ˆ –‘–ƒŽ ‘”–Š‘’Š‘•’Šƒ–‡
ƒ† ȾǦ ‘š‹†‹œ‡† ȋ‹‰—”‡ ͷȌǤ ”‘ –Š‡ –‘–ƒŽ ‘”–Š‘’Š‘•’Šƒ–‡ …‘…‡–”ƒ–‹‘ǡ ™‡ –Š‡
•—„–”ƒ…–‡† –Š‡ ˆ”ƒ…–‹‘ ‰‡‡”ƒ–‡† „› ‡… ȋ…‘””‡•’‘†‹‰ –‘ –Š‡ †‡…”‡ƒ•‡ ‘ˆ ȾǦ
…‘…‡–”ƒ–‹‘Ȍǡƒ†™‡ƒ……‘”†‹‰Ž›‘„–ƒ‹‡†–Š‡…‘…‡–”ƒ–‹‘‘ˆ’›”‘’Š‘•’Šƒ–‡”‡Ž‡ƒ•‡†„›
Ƚ” ȋ‹‰—”‡ ͷȌǤ —”’‹•‹‰Ž›ǡ Ƚ” †‹† ’‡”ˆ‘” ‡š–‡•‹‘ ƒ……‘”†‹‰ –‘ ƒ œ‡”‘Ǧ‘”†‡”
‹‡–‹…• ˆ‘” –Š‡ ˆ‹”•– ͸Ͳ ‹ ‘ˆ ”‡ƒ…–‹‘ –‹‡ ȋ‹‰—”‡ ͷȌǡ ƒ† –Š‡ ƒ‰‹–—†‡ ‘ˆ –Š‹• ƒ…–‹˜‹–›
™ƒ• •‹‰‹ˆ‹…ƒ–Ž› Š‹‰Š‡” –Šƒ –Š‡ ‘Ž ƒ…–‹˜‹–› ‘„•‡”˜‡† ƒ– –Š‡ ‡š’‡•‡ ‘ˆ ƒ †• ‘–
”‡“—‹”‹‰”‡…‘„‹ƒ–‹‘–‘„‡‡š–‡†‡†ȋ…ˆǤ‹‰—”‡•ʹƒ†͸ȌǤ–Š‡…‘–”ƒ”›ǡ™Š‡Ƚ…
™ƒ• ƒ••ƒ›‡† —†‡” –Š‡ •ƒ‡ …‘†‹–‹‘• ȋ‹‰—”‡• ͷǦͷȌǡ –Š‡ ƒ…–‹˜‹–› ‘ˆ –Š‹• ‘Ž ™ƒ•
…Ž‡ƒ”Ž› —…Š Ž‘™‡” –Šƒ –Š‡ ‡š–‡•‹‘ ƒ…–‹˜‹–› ‘„•‡”˜‡† ‹ –Š‡ ƒ„•‡…‡ ‘ˆ ‡… ȋ…ˆǤ
‹‰—”‡• ʹ ƒ† ͸ȌǤ ‘”‡‘˜‡”ǡ ƒ …Ž‡ƒ” Žƒ‰ ™ƒ• ‘„•‡”˜‡† „‡ˆ‘”‡ –Š‡ ‘Ž ƒ…–‹˜‹–› ‘ˆ Ƚ…
‡–‡”‡†œ‡”‘Ǧ‘”†‡”‹‡–‹…•ȋ‹‰—”‡ͷȌǡƒ†–Š‹•‹‡–‹…”‡‰‹‡™ƒ•ƒ‹–ƒ‹‡†ˆ‘”ƒ„‘—–ʹͲ
‹ ‘Ž› ȋ‹‰—”‡ ͷȌǤ ˜‡”ƒŽŽǡ –Š‡•‡ ‘„•‡”˜ƒ–‹‘• •—‰‰‡•– –Šƒ– Ƚ” ‹• •–‹—Žƒ–‡† „› E. coli
‡…ǡ ƒ† –Šƒ– —†‡” –Š‡ …‘†‹–‹‘• ‘ˆ –Š‡ Ǧ ƒ••ƒ› Ƚ” ‹• ‘”‡ ’”‘…‡••‹˜‡ –Šƒ Ƚ…Ǥ
……‘”†‹‰Ž›ǡ ™‡ ™ƒ–‡† –‘ –‡•– –Š‡ ‘Ž ƒ…–‹˜‹–› ‘ˆ „‘–Š Ƚ” ƒ† Ƚ… —†‡” …‘†‹–‹‘•
”‡“—‹”‹‰”‡…‘„‹ƒ–‹‘ǡ„—–‹–Š‡ƒ„•‡…‡‘ˆ„‘–Š‡…ƒ†ȋƒ†–Š‡”‡ˆ‘”‡†‡˜‘‹†‘ˆ
ǡǡǡƒ†ȾǦȌǤ›–Š‹•‡ƒ•ǡ™‡™ƒ–‡†–‘ƒ••ƒ›–Š‡‹–”‹•‹…”‡…‘„‹ƒ–‹‘
ƒ…‹–‹˜‹–›ǡ‹ˆƒ–ƒŽŽǡ‘ˆȽ”ƒ†Ƚ…Ǥ—”’”‹•‹‰Ž›ƒ‰ƒ‹ǡ™Š‹Ž‡Ƚ…™ƒ•…Ž‡ƒ”Ž›ƒ„Ž‡–‘…ƒ–ƒŽ›œ‡–Š‡
”‡…‘„‹ƒ–‹‘ƒ†‡š–‡•‹‘‘ˆǡƒŽ„‡‹–ƒ–ƒ‘†‡”ƒ–‡”ƒ–‡ǡȽ”™ƒ•‹…ƒ’ƒ„Ž‡–‘’‡”ˆ‘”
–Š‡•‡ –ƒ••ǡ ‘•– Ž‹‡Ž› „‡…ƒ—•‡ ‘ˆ ‹–• ‹‡ˆˆ‹…‹‡…› ‹ ”‡…‘„‹ƒ–‹‘ ƒ…–‹˜‹–› ȋ‹‰—”‡ ͸ǡ ƒ†
—’’Ž‡‡–ƒ”›‹‰—”‡ͶȌǤ
ͳͷ
RecombinationǦdependentpolymeraseactivityofȽDroverexpressedinsolubleform
‘ ˆ—”–Š‡” ‹˜‡•–‹‰ƒ–‡ –Š‡ ‡…Ǧ†‡’‡†‡…‡ ‘ˆ Ƚ” ’‘Ž›‡”ƒ•‡ ƒ…–‹˜‹–›ǡ ™‡ ’‡”ˆ‘”‡†
”‡…‘„‹ƒ–‹‘Ǧ†‡’‡†‡– ‡š–‡•‹‘ ƒ••ƒ›• —•‹‰ ’ƒ”–‹ƒŽŽ› ’—”‹ˆ‹‡† Ƚ” ‘˜‡”‡š’”‡••‡†
‹•‘Ž—„Ž‡ˆ‘”ǤŠ‡–Š‡’‘Ž›‡”ƒ•‡ƒ…–‹˜‹–›‘ˆȽ”™ƒ•–‡•–‡†—•‹‰–Š‡Ǧƒ••ƒ›ȋ‹
–Š‡ ’”‡•‡…‡ ‘ˆ ‡…Ȍǡ ™‡ ‘„•‡”˜‡† ƒ •‹‰‹ˆ‹…ƒ– ‡š–‡•‹‘ǡ ƒ– –Š‡ ‡š’‡•‡ ‘ˆ †ǡ ‘ˆ –Š‡
†• ‰‡‡”ƒ–‡† „› ‡… ȋ‹‰—”‡ ͹ȌǤ  –Š‡ …‘–”ƒ”›ǡ ‘ ’‘Ž›‡”ƒ•‡ ƒ…–‹˜‹–› ™ƒ•
†‡–‡…–‡†™Š‡Ƚ”™ƒ•ƒ††‡†–‘ƒ”‡ƒ…–‹‘‹š–—”‡†‡˜‘‹†‘ˆ‡…ǡƒ†…‘–ƒ‹‹‰–Š‡–™‘
•–‘„‡”‡…‘„‹‡†ƒ††ȋ—’’Ž‡‡–ƒ”›‹‰—”‡ͷȌǤŠ‡•‡‘„•‡”˜ƒ–‹‘•“—ƒŽ‹–ƒ–‹˜‡Ž›
ƒ‰”‡‡™‹–Š–Š‘•‡‘„–ƒ‹‡†—•‹‰”‡ˆ‘Ž†‡†Ƚ”‹•‘Žƒ–‡†ƒ†’—”‹ˆ‹‡†ˆ”‘‹…Ž—•‹‘„‘†‹‡•Ǥ
ͳ͸
DISCUSSION
Š‡ ’—”‹ˆ‹…ƒ–‹‘ ‘ˆ –Š‡ Deinococcus radiodurans ‘Ž›‡”ƒ•‡ Ƚ •—„—‹– ȋȽ”Ȍ ‹•
†‡•…”‹„‡†Š‡”‡ˆ‘”–Š‡ˆ‹”•––‹‡Ǥƒ–Š‡”•—’”‹•‹‰Ž›ǡ™Š‡‘˜‡”‡š’”‡••‡†‹•‘Ž—„Ž‡ˆ‘”ǡȽ”
ˆ‡ƒ–—”‡†ƒ•–”‘‰„‹†‹‰–‘–Š‡‘Ž›‡”ƒ•‡‘ˆ–Š‡Š‘•–ǡ‹Ǥ‡ǤE.coliǤ……‘”†‹‰Ž›ǡ™‡™‡”‡
ƒ„Ž‡ –‘ ’ƒ”–‹ƒŽŽ› ’—”‹ˆ› ƒ–‹˜‡ Ƚ” ƒ••‘…‹ƒ–‡† –‘ E. coli ‘Žǡ ƒ† ™‡ ‘„•‡”˜‡† –Šƒ– –Š‡
†‹••‘…‹ƒ–‹‘ ‘ˆ –Š‹• …‘’Ž‡š †‘‡• ‘– ‘……—” —†‡” †‹ˆˆ‡”‡– …‘†‹–‹‘•ǡ ‡Ǥ‰Ǥ ‰‡Ž ˆ‹Ž–”ƒ–‹‘ ‘”
Š‹‰Š …‘…‡–”ƒ–‹‘• ‘ˆ ȋ†ƒ–ƒ ‘– •Š‘™ȌǤ Š‡–Š‡” ‘” ‘– Ƚ” †‘‡• ƒŽ•‘ ˆ‡ƒ–—”‡ Š‹‰Š
ƒˆˆ‹‹–›ˆ‘”D.radiodurans‘Ž”‡ƒ‹•–‘„‡ƒ•…‡”–ƒ‹‡†Ǥ–Š‡’”‡•‡–™‘”ǡ™‡—•‡†
–Š‡ Ƚ”Ǧ ‘Ž …‘’Ž‡š ˆ‘” “—ƒŽ‹–ƒ–‹˜‡ ƒ…–‹˜‹–› ƒ••ƒ›• ‘Ž›ǡ †‡•‹‰‡† –‘ ƒ˜‘‹† ƒ›
‹–‡”ˆ‡”‡…‡ „› ‘Ž ‘ –Š‡ ‘„•‡”˜‡† ƒ…–‹˜‹–‹‡•Ǥ – •Š‘—Ž† „‡ ‹†‡‡† ‘–‡† –Šƒ– ™‡
’‡”ˆ‘”‡† ‘Ž ƒ…–‹˜‹–› ƒ••ƒ›• ”‡Ž›‹‰ ‘ † ƒ• –Š‡ —‹“—‡ —…Ž‡‘–‹†‡ •—„•–”ƒ–‡Ǥ ‘
‘„–ƒ‹Š‘‘‰‡‡‘—•Ƚ”ˆ”‡‡‘ˆ‘Žǡ™‡‘˜‡”‡š’”‡••‡†Ƚ”ƒ•‹…Ž—•‹‘„‘†‹‡•ǡƒ†™‡
™‡”‡•—……‡••ˆ—Ž‹–Š‡”‡ˆ‘Ž†‹‰ƒ†’—”‹ˆ‹…ƒ–‹‘‘ˆ–Š‡–ƒ”‰‡–’”‘–‡‹Ǥ
Š‡ …‘’ƒ”‡† –‘ E. coli ‘Ž›‡”ƒ•‡ Ƚ •—„—‹– ȋȽ…Ȍǡ Ƚ” •Š‘™‡† “—‹–‡ †‹ˆˆ‡”‡–
‹‡–‹…’”‘’‡”–‹‡•Ǥ†‡”•–ƒ†ƒ”†…‘†‹–‹‘•ȋͷ‰ŽʹǡͳͲͲɊ†ǡŽ‘™‹‘‹…•–”‡‰–ŠȌ
ƒ† ‹ –Š‡ ’”‡•‡…‡ ‘ˆ ͳ Ɋ ͶͲ‡” –‡’Žƒ–‡ ǡ Ƚ” ™ƒ• ‹†‡‡† ˆ‘—† –‘ …ƒ–ƒŽ›œ‡ ‡š–‡•‹‘™‹–Š—…ŠŽ‘™‡”‡ˆˆ‹…‹‡…›–ŠƒȽ…Ǥ–‡”‡•–‹‰Ž›ǡ™Š‡–Š‡‹‘‹…•–”‡‰–Š‘ˆ–Š‡
ƒ••ƒ›‹š–—”‡™ƒ•‹…”‡ƒ•‡†ǡ–Š‡…ƒ–ƒŽ›–‹…ƒ…–‹‘‘ˆȽ”ƒ†Ƚ…™‡”‡ˆ‘—†–‘‹…”‡ƒ•‡ƒ†
†‡…”‡ƒ•‡ǡ ”‡•’‡…–‹˜‡Ž›Ǥ ‘”‡‘˜‡”ǡ –Š‡ ƒ††‹–‹‘ ‘ˆ ʹΪ –‘ –Š‡ ”‡ƒ…–‹‘ ‹Ž‹‡— –”‹‰‰‡”‡† ƒ
•–”‘‰ ‹…”‡ƒ•‡ ‹ Ƚ” ƒ…–‹˜‹–›ǡ ƒ† †‹† …‘””‡•’‘† –‘ ƒ ‘†‡•–ǡ ‹ˆ ƒ– ƒŽŽǡ ‡ˆˆ‡…– –‘™ƒ”†• –Š‡
ƒ…–‹‘‘ˆȽ…Ǥ–‹•‹–‡”‡•–‹‰–‘‘–‡–Šƒ–ɀǦ‹””ƒ†‹ƒ–‹‘‹†—…‡•ƒ‰‡‡”ƒŽ‹…”‡ƒ•‡‘ˆ…‡ŽŽ—Žƒ”
‹‘‹… •–”‡‰–Šǡ ƒ† –Šƒ– –Š‡ ƒ……——Žƒ–‹‘ ‘ˆ ʹΪ ‹…”‡ƒ•‡• –Š‡ ”ƒ†‹‘”‡•‹•–ƒ…‡ ‘ˆ D.
radioduransȏͷ͵ȐǤŠ‡”‡ˆ‘”‡ǡ‘—”‘„•‡”˜ƒ–‹‘••—‰‰‡•––Šƒ––Š‡ƒ…–‹˜‹–›‘ˆȽ”‹•”‡‰—Žƒ–‡†„›
–Š‡…‘†‹–‹‘•ƒ”‹•‹‰‹…‡ŽŽ•—’‘‡š’‘•—”‡–‘ɀǦ”ƒ›•Ǥ’ƒ”–‹…—Žƒ”ǡ–Š‡’‘•‹–‹˜‡”‡•’‘•‡‘ˆ
ͳ͹
Ƚ”–‘ʹΪ”‡’‘”–‡†Š‡”‡ƒ‰”‡‡•™‹–Š’”‡˜‹‘—••–—†‹‡•…‘…‡”‹‰D.radiodurans‘Žƒ†
‘ŽǤ–™ƒ•‹†‡‡††‡‘•–”ƒ–‡†–Šƒ–ʹΪ‹•‡••‡–‹ƒŽˆ‘”–Š‡ƒ…–‹˜‹–›‘ˆD.radiodurans‘Ž
ȏ͵͹Ȑǡ ƒ† ‹• ”‡“—‹”‡† –‘ …‘ˆ‡” …‘’‡–‡‡…‡ ‹ Ž‡•‹‘ „›’ƒ•• –‘ ‘Ž ȏͶʹȐǤ
……‘”†‹‰Ž›ǡ–Š‡‘Ž•‘ˆD.radiodurans•Šƒ”‡ƒ•–”‘‰†‡’‡†‡…‡‘ƒ‰ƒ‡•‡‘ˆ–Š‡‹”
ƒ…–‹˜‹–‹‡•ǡ•—‰‰‡•–‹‰ƒ‹…‡…‘””‡Žƒ–‹‘™‹–Š–Š‡Š‹‰Š…‘–‡–‘ˆƒ‰ƒ‡•‡™Š‹…Š‹•—•—ƒŽŽ›
†‡–‡…–‡† ‹ ’‘•–Ǧ‹””ƒ–‡† …‡ŽŽ• ‘ˆ –Š‹• ‘”‰ƒ‹•Ǥ ‘™‡˜‡”ǡ ‹– •Š‘—Ž† ƒŽ•‘ „‡ …‘•‹†‡”‡† –Šƒ–
‘–Š‡” ‘Ž•ǡ ‹•‘Žƒ–‡† ˆ”‘ ”ƒ†‹ƒ–‹‘Ǧ•‡•‹–‹˜‡ ‘”‰ƒ‹••ǡ †‡’‡† ‘ ʹΪ ˆ‘” –Š‡‹”
ƒ…–‹˜‹–›ǡ‡Ǥ‰Ǥ–Š‡Š—ƒɇ‘ŽȏͷͶȐǤ
Š‡…‘•‹†‡”‹‰–Šƒ––Š‡’ƒ–Š™ƒ›‘ˆ‰‡‘‡”‡…‘•–‹–—–‹‘‹••–”‹…–Ž›†‡’‡†‡–‘
Ƚ”ǡ ‘‡ ‹‰Š– ‡š’‡…– –Šƒ– –Š‹• ‘Ž›‡”ƒ•‡ ˆ‡ƒ–—”‡• ‹–”‹•‹… ”‡…‘„‹ƒ•‡ ƒ…–‹˜‹–›Ǥ
‘™‡˜‡”ǡ Ƚ” ™ƒ• ˆ‘—† –‘ „‡ ‹…ƒ’ƒ„Ž‡ ‘ˆ ”‡…‘„‹ƒ–‹‘Ǧ†‡’‡†‡– ’‘Ž›‡”ƒ•‡
ƒ…–‹˜‹–›ǡ ‹†‡’‡†‡–Ž› ‘ˆ –Š‡ •–”ƒ–‡‰› —•‡† –‘ ‘˜‡”‡š’”‡•• Ƚ” ‹ E. coli ȋ—’’Ž‡‡–ƒ”›
‹‰—”‡•Ͷƒ†ͷȌǤ–Š‡…‘–”ƒ”›ǡ–Š‹•ƒ…–‹˜‹–›™ƒ•‡š…Ž—•‹˜‡Ž›†‡–‡…–‡†‹–Š‡’”‡•‡…‡‘ˆ
„‘–Š‡…ƒ†Ƚ”ǤŠ‹•‹•‹•Šƒ”’…‘–”ƒ•–™‹–Š–Š‡…ƒ–ƒŽ›–‹…’”‘’‡”–‹‡•‘ˆȽ…ǡ™Š‹…Š™ƒ•
ˆ‘—† …‘’‡–‡– ‹ ”‡…‘„‹ƒ–‹‘Ǧ†‡’‡†‡– ’‘Ž›‡”ƒ•‡ ƒ…–‹˜‹–› ‹ –Š‡ ƒ„•‡…‡ ‘ˆ
‡…ȋ—’’Ž‡‡–ƒ”›‹‰—”‡•Ͷƒ†ͷȌǤ–•Š‘—Ž†ƒŽ•‘„‡‘–‡†–Šƒ––Š‡ƒ…–‹˜‹–›‘ˆȽ”‹–Š‡
’”‡•‡…‡ ‘ˆ ‡… •‹‰‹ˆ‹…ƒ–Ž› ‡š…‡‡†• –Š‡ ‡š–‡•‹‘ ”ƒ–‡ ‘„•‡”˜‡† ™‹–Š ƒ ”‡…‘„‹ƒ–‹‘Ǧ
‹†‡’‡†‡–•—„•–”ƒ–‡ȋ…ˆǤ‹‰—”‡•͸ƒ†ʹȌǡ•—‰‰‡•–‹‰ƒƒ…–‹˜ƒ–‹‘‘ˆȽ”„›‡…Ǥ
Š‹•‡˜‹†‡…‡ƒ‰”‡‡•™‹–Š–Š‡ˆ‹†‹‰–Šƒ–‰‡‘‡”‡’Ž‹…ƒ–‹‘‹D.radiodurans‡…Ǧ•–”ƒ‹•
‹••Ž‘™‡”™Š‡…‘’ƒ”‡†™‹–Š–Šƒ–‘„•‡”˜‡†‹–Š‡™‹Ž†Ǧ–›’‡…‘—–‡”’ƒ”–ȏ͵͸ȐǤ‘˜‡”•‡Ž›ǡ
†‡Ž‡–‹‘‘ˆ‡…†‘‡•‘–•‹‰‹ˆ‹…ƒ–Ž›ƒˆˆ‡…––Š‡‰”‘™–Š”ƒ–‡‘ˆE.coliȏͷͷȐǤ‘”‡‘˜‡”ǡ‹–™ƒ•
†‡‘•–”ƒ–‡† –Šƒ– –Š‡ ‘˜‡”‡š’”‡••‹‘ ‘ˆ ‡… †‡…”‡ƒ•‡• –Š‡ ”ƒ–‡ ‘ˆ ”‡’Ž‹…ƒ–‹‘ ˆ‘”
’”‘‰”‡••‹‘‹E.coliȏʹͻȐǡ’”‡•—ƒ„Ž›„›‹Š‹„‹–‹‰Ƚ…”‡’Ž‹•‘‡•ȏʹͺȐǤ
‡‘„•‡”˜‡†–Šƒ––Š‡”ƒ–‡ƒ†–Š‡‡š–‡–‘ˆŠ›†”‘Ž›•‹•†‹ˆˆ‡”‹ˆȽ”‘”Ƚ…™‡”‡’”‡•‡–
‹‡…Ǧ†‡’‡†‡–‡š–‡•‹‘ƒ••ƒ›•Ǥ—”‹‰–Š‡ˆ‹”•–͸ͲǦ͹Ͳ‹ƒˆ–‡””‡ƒ…–‹‘••–ƒ”–‡†ǡ
ͳͺ
™‡ †‹† ‹†‡‡† ƒ•…‡”–ƒ‹ –Šƒ–ǣ ‹Ȍ ™Š‡ Ƚ” ™ƒ• ’”‡•‡–ǡ ͷ Ɋ † ™ƒ• ‹…‘”’‘”ƒ–‡† ‹–‘
ǡƒ†–Š‡Š›†”‘Ž›•‹•‘ˆͳͲǦͳͷɊ™ƒ•…‘…‘‹–ƒ–Ž›†‡–‡…–‡†ȋ‹‰—”‡ͺȌǢ‹‹Ȍ™Š‡
Ƚ…†‹†…ƒ–ƒŽ›œ‡–Š‡‡š–‡•‹‘‘ˆƒ––Š‡‡š’‡•‡‘ˆͶǦͷɊ†ǡ‡…†‹†Š›†”‘Ž›œ‡ʹͲǦ
͵ͲɊȋ‹‰—”‡ͺȌǤŠ‡•‡ˆ‹†‹‰••—‰‰‡•––Šƒ–Ƚ”†‘‡•ƒ••‹•–‡…‹–Š‡”‡…‘„‹ƒ–‹‘
”‡ƒ…–‹‘ǡŽ‘™‡”‹‰–Š‡ƒ‘—–‘ˆ‡…‡••ƒ”›–‘•—•–ƒ‹•–”ƒ†‡š…Šƒ‰‡ƒ†ǡŠ‡…‡ǡ
‡š–‡•‹‘Ǥ–‡”‡•–‹‰Ž›ǡ–Š‡‡‡”‰‡–‹…•‘ˆ‡…Ǧ‡†‹ƒ–‡†”‡…‘„‹ƒ–‹‘™ƒ•ˆ‹‡Ž›†‹••‡…–‡†
„› ƒ •‡”‹‡• ‘ˆ ‡Ž‡‰ƒ– •–—†‹‡•Ǥ ‹”•–ǡ ‹– ™ƒ• †‡‘•–”ƒ–‡† –Šƒ– –Š‡ ”ƒ–‡ ‘ˆ Š›†”‘Ž›•‹•
–”‹‰‰‡”‡†„›–Š‡ƒ••‘…‹ƒ–‹‘‘ˆ‡…–‘•••Ž‘™•†‘™—’‘‹‹–‹ƒ–‹‘‘ˆ•–”ƒ†‡š…Šƒ‰‡
™‹–Š †• ȏͷ͸ȐǤ —”–Š‡”ǡ ‹– ™ƒ• ”‡…‘‰‹œ‡† –Šƒ–ǣ ‹Ȍ –Š‡ „‹†‹‰ ‘ˆ ‘” ‘ˆ ƒƒŽ‘‰•
…‘˜‡”–• ‡… ‹ƒ…–‹˜‡ ˆ‹Žƒ‡–• –‘ ƒ ƒ…–‹˜‡ •–ƒ–‡ ȏͷ͹ȐǢ ‹‹Ȍ Š›†”‘Ž›•‹• ƒ†
”‡…‘„‹ƒ–‹‘ƒŽ„”ƒ…Š‹‰”ƒ–‹‘ƒ”‡…‘…‡”–‡†”‡ƒ…–‹‘•ȏͷͺȐǤ‘”‡‘˜‡”ǡ‹–™ƒ•ƒŽ•‘•Š‘™
–Šƒ– Š›†”‘Ž›•‹• ‹†—…‡• ƒ …‘ˆ‘”ƒ–‹‘ƒŽ –”ƒ•‹–‹‘ ‘ˆ ‡… Ǧ–‡”‹—•ǡ ƒ† –Šƒ– –Š‹•
–”ƒ•‹–‹‘ …‘””‡•’‘†• –‘ –Š‡ …‘˜‡”•‹‘ ‘ˆ ‹ƒ…–‹˜‡ ‡… ˆ‹Žƒ‡–• –‘ –Š‡‹” ƒ…–‹˜‡
…‘—–‡”’ƒ”– ȏͷͻǡ͸ͲȐǤ ‹ƒŽŽ›ǡ †‹ˆˆ‡”‡– •–ƒ–‡• ‘ˆ ‡… ˆ‹Žƒ‡–• ™‡”‡ ‹†‡–‹ˆ‹‡† ȏ͸ͳȐǤ  –Š‡
ƒ„•‡…‡‘ˆǡ‡…‹•‹–Š‡•–ƒ–‡ƒ†‹•…‘’‡–‡–‹„‹†‹‰••ȏ͸ͳȐǤŠ‡‹•
’”‡•‡–ǡ –Š‡ ”‡…‘„‹ƒ•‡ •™‹–…Š‡• –‘ –Š‡ ȋƒ…–‹˜ƒ–‡†Ȍ •–ƒ–‡ǡ ƒ† –Š‡ „‹†‹‰ ‘ˆ ••
–”‹‰‰‡”•Š›†”‘Ž›•‹•ǤŠ‡•–ƒ–‡…ƒ„‡ˆ—”–Š‡”…Šƒ”ƒ…–‡”‹œ‡†‹–™‘•—„Ǧ•–ƒ–‡•ǡ†‡‘–‡†
…ȋ…Ž‘•‡†Ȍƒ†‘ȋ‘’‡Ȍǡ–‘‹†‹…ƒ–‡‹…‘’‡–‡…‡ƒ†…‘’‡–‡…‡‹’ƒ‹”‹‰”‡ƒ…–‹‘•ǡ
”‡•’‡…–‹˜‡Ž› ȏ͸ͳȐǤ Š‡ –”ƒ•‹–‹‘ ˆ”‘ –Š‡ … –‘ –Š‡ ‘ •–ƒ–‡ ™ƒ• ˆ‘—† –‘ †‡’‡† ‘ ‰ʹΪ
…‘…‡–”ƒ–‹‘ȏ͸ʹȐǤ–Š‡’”‡•‡…‡‘ˆƒ’ƒ‹”‹‰•—„•–”ƒ–‡ǡ‡…•™‹–…Š‡•ˆ”‘–Š‡‘–‘–Š‡
•–ƒ–‡ǡƒ†–Š‹•–”ƒ•‹–‹‘‹•’ƒ”ƒŽŽ‡Ž‡†„›ƒ†”‡…”‡ƒ•‡ȋƒ„‘—–͵ͲΨȌ‘ˆ–Š‡Š›†”‘Ž›•‹•”ƒ–‡
ȏ͸ͳȐǤ……‘”†‹‰–‘–Š‡•‡‘„•‡”˜ƒ–‹‘•ǡ™‡’”‘’‘•‡–Šƒ–Ƚ”ˆƒ˜‘—”•–Š‡–”ƒ•‹–‹‘ˆ”‘–Š‡‘
–‘ –Š‡ •–ƒ–‡ ‘ˆ ‡…ǡ –Š‡”‡ˆ‘”‡ Ž‹‹–‹‰ –Š‡ ƒ‘—– ‘ˆ ‡…‡••ƒ”› –‘ ƒ……‘’Ž‹•Š
”‡…‘„‹ƒ–‹‘Ǥ  ’ƒ”–‹…—Žƒ”ǡ ‹ –Š‡ ’”‡•‡…‡ ‘ˆ Ƚ” –Š‡ …‘•—‡† ’‡” —…Ž‡‘–‹†‡
ͳͻ
‹…‘”’‘”ƒ–‡†‹™ƒ•‡“—ƒŽ–‘ƒ„‘—–ʹǡ™Š‹Ž‡‹–Š‡’”‡•‡…‡‘ˆȽ…–Š‹•”ƒ–‹‘‹•‘”‡–Šƒ
†‘—„Ž‡ȋ‹‰—”‡ͺȌǤ
˜‡”ƒŽŽǡ™‡Šƒ˜‡•Š‘™Š‡”‡–Šƒ–•‘‡‘ˆ–Š‡…ƒ–ƒŽ›–‹…’”‘’‡”–‹‡•‘ˆȽ”ƒ”‡‹•Šƒ”’…‘–”ƒ•–
™‹–Š–Š‘•‡‘ˆȽ…ǣ‹ȌȽ”‹••–‹—Žƒ–‡†„›Š‹‰Š‹‘‹…•–”‡‰–Šǡ™Š‹Ž‡Ƚ…‹••–”‘‰Ž›‹Š‹„‹–‡†
—†‡”–Š‡•ƒ‡…‘†‹–‹‘•Ǣ‹‹Ȍ•‹‹Žƒ”Ž›–‘‘–Š‡”‘Ž•‘ˆD.radioduransǡȽ”‹•ƒ…–‹˜ƒ–‡†
„›ʹΪǡ™Š‹Ž‡Ƚ…ƒ…–‹‘‹•‘–•‹‰‹ˆ‹…ƒ–Ž›ƒˆˆ‡…–‡†„›ƒ‰ƒ‡•‡Ǣ‹‹‹Ȍ…‘–”ƒ”›–‘Ƚ…ǡȽ”
‹• ƒ…–‹˜ƒ–‡† „› ‡…ǡ ‹ –Š‡ ƒ„•‡…‡ ‘ˆ ™Š‹…Š Ƚ” ‹• ‘– ƒ„Ž‡ –‘ …ƒ–ƒŽ›œ‡ ”‡…‘„‹ƒ–‹‘Ǧ
†‡’‡†‡–‡š–‡•‹‘Ǥ‡ƒ”ƒ„Ž›ǡ–Š‡ƒ…–‹‘•‘ˆE.coliƒ†D.radiodurans‡…’”‘–‡‹•
™‡”‡”‡’‘”–‡†–‘†‹ˆˆ‡”•—„•–ƒ–‹ƒŽŽ›ȏ͸͵ǡ͸ͶȐǤŠ‹Ž‡E.coli‡…†‘‡•‹†‡‡†’”‡ˆ‡”••ƒ•
•—„•–”ƒ–‡ȋƒ•ƒŽ•‘•Š‘™Š‡”‡ǡ•‡‡‹‰—”‡͵Ȍǡ–Š‡D.radiodurans”‡…‘„‹ƒ•‡™ƒ•”‡’‘”–‡†–‘
„‹† ‘”‡ ‡ˆˆ‹…‹‡–Ž› †•Ǥ ……‘”†‹‰Ž›ǡ ˆ—–—”‡ ™‘” ™‹ŽŽ „‡ †‡˜‘–‡† –‘ ‹˜‡•–‹‰ƒ–‡ –Š‡
…ƒ–ƒŽ›–‹… ƒ…–‹‘ ‘ˆ Ƚ” ‹ ”‡…‘„‹ƒ–‹‘Ǧ†‡’‡†‡– ’‘Ž›‡”ƒ•‡ ƒ…–‹˜‹–› ƒ••ƒ›•
’‡”ˆ‘”‡†‹–Š‡’”‡•‡…‡‘ˆD.radiodurans‡…’”‘–‡‹Ǥ
ʹͲ
REFERENCES
ͳ
‘›…‡ǡ Ǥ Ǥ ƒ† –‡‹–œǡ Ǥ Ǥ ȋͳͻͻͶȌ —…–‹‘ ƒ† •–”—…–—”‡ ”‡Žƒ–‹‘•Š‹’• ‹ ’‘Ž›‡”ƒ•‡•Ǥ—Ǥ‡˜Ǥ‹‘…Š‡Ǥ63ǡ͹͹͹ǦͺʹʹǤ
ʹ
…‡”›ǡ Ǥ Ǥ ȋʹͲͳͳȌ ”‡’Ž‹…ƒ•‡• ˆ”‘ ƒ „ƒ…–‡”‹ƒŽ ’‡”•’‡…–‹˜‡Ǥ —Ǥ ‡˜Ǥ
‹‘…Š‡Ǥ80ǡͶͲ͵ǦͶ͵͸Ǥ
͵
‡”‡ǡǤǡ
”‹†Ž‡›ǡǤǤǤƒ†‘›…‡ǡǤǤȋʹͲͳͳȌ‹•–‹…–”‘Ž‡•‘ˆ–Š‡ƒ…–‹˜‡Ǧ•‹–‡
‰ʹڎ‹‰ƒ†•ǡ•’ͺͺʹƒ†•’͹Ͳͷǡ‘ˆ’‘Ž›‡”ƒ•‡ȋŽ‡‘™ˆ”ƒ‰‡–Ȍ†—”‹‰–Š‡
’”‡…Š‡‹•–”›…‘ˆ‘”ƒ–‹‘ƒŽ–”ƒ•‹–‹‘•ǤǤ‹‘ŽŠ‡Ǥ286ǡ͵͹ͷͷǦ͵͹͸͸Ǥ
Ͷ
‡ˆ–‡”ǡǤǤǡ‹”‘–ƒǡǤǡ‘”„‡”‰ǡǤǡ‡…Š•Ž‡”ǡǤǤƒ†ƒ”‘—šǡǤȋͳͻ͹ͳȌƒŽ›•‹•
‘ˆ ’‘Ž›‡”ƒ•‡• ƒ† ‹ —–ƒ–• ‘ˆ Escherichia coli –Š‡”‘•‡•‹–‹˜‡ ˆ‘”
•›–Š‡•‹•Ǥ”‘…Ǥƒ–ŽǤ…ƒ†Ǥ…‹Ǥ68ǡ͵ͳͷͲǦ͵ͳͷ͵Ǥ
ͷ
ò••Ž‡‹ǡ Ǥǡ ‡”†ǡ Ǥǡ ‘Š‘‡ˆˆ‡”ǡ Ǥ ƒ† …ŠƒŽŽ‡”ǡ Ǥ ȋͳͻ͹ͳȌ —…–‹‘ ‘ˆ ’‘Ž›‡”ƒ•‡‹”‡’Ž‹…ƒ–‹‘Ǥƒ–—”‡‡™‹‘ŽǤ234ǡʹͺͷǦʹͺ͸Ǥ
͸
‘”„‡”‰ǡ Ǥ ƒ† ‡ˆ–‡”ǡ Ǥ Ǥ ȋͳͻ͹ʹȌ ‡‘š›”‹„‘—…Ž‡‹… ƒ…‹† •›–Š‡•‹• ‹ …‡ŽŽǦˆ”‡‡
‡š–”ƒ…–•Ǥ Ǥ —”‹ˆ‹…ƒ–‹‘ ƒ† …ƒ–ƒŽ›–‹… ’”‘’‡”–‹‡• ‘ˆ †‡‘š›”‹„‘—…Ž‡‹… ƒ…‹†
’‘Ž›‡”ƒ•‡ǤǤ‹‘ŽǤŠ‡Ǥ247ǡͷ͵͸ͻǦͷ͵͹ͷǤ
͹
‡ —…‹ƒǡ Ǥ ƒ† ƒ‹”•ǡ Ǥ ȋͳͻ͸ͻȌ •‘Žƒ–‹‘ ‘ˆ ƒ E. coli •–”ƒ‹ ™‹–Š ƒ —–ƒ–‹‘
ƒˆˆ‡…–‹‰’‘Ž›‡”ƒ•‡Ǥƒ–—”‡224ǡͳͳ͸ͶǦͳͳ͸͸Ǥ
ͺ
‘›…‡ǡǤǤƒ†
”‹†Ž‡›ǡǤǤȋͳͻͺͶȌ‡–Š‘†ˆ‘”†‡–‡”‹‹‰™Š‡–Š‡”ƒ‰‡‡‘ˆ
Escherichiacoli‹•‡••‡–‹ƒŽǣƒ’’Ž‹…ƒ–‹‘–‘–Š‡polA‰‡‡ǤǤƒ…–‡”‹‘ŽǤ158ǡ͸͵͸Ǧ͸Ͷ͵Ǥ
ͻ
‹’’‡”•ǡǤȋͳͻ͹ͲȌ’‘Ž›‡”ƒ•‡Ǥƒ–—”‡228ǡͳͲͷͲǦͳͲͷ͵Ǥ
ͳͲ
‹ǡ Ǥǡ ƒ‡Šƒ—–Ǧ‹…Šƒ‡Žǡ Ǥǡ ƒƒ†ƒǡ Ǥǡ ƒƒ‘–‘ǡ Ǥǡ ƒ–•—‹ǡ Ǥǡ ‘ˆ—‹ǡ Ǥǡ
‘Š‹ǡǤƒ†Š‘”‹ǡǤȋͳͻͻ͹Ȍ—Ž–‹’Ž‡’ƒ–Š™ƒ›•ˆ‘”Ǧ‹†—…‡†—–ƒ‰‡‡•‹•
‹ Escherichia coliǣ ƒ ‘˜‡”‡š’”‡••‹‘ ‘ˆ dinB/dinP ”‡•—Ž–• ‹ •–”‘‰Ž› ‡Šƒ…‹‰
ʹͳ
—–ƒ‰‡‡•‹•‹–Š‡ƒ„•‡…‡‘ˆƒ›‡š‘‰‡‘—•–”‡ƒ–‡––‘†ƒƒ‰‡Ǥ”‘…Ǥƒ–ŽǤ
…ƒ†Ǥ…‹Ǥ94ǡͳ͵͹ͻʹǦͳ͵͹ͻ͹Ǥ
ͳͳ
‡—˜‡ǡǤǤǡ‘‡”ǡ
Ǥƒ†‹˜‡ŠǡǤȋͳͻͻͺȌŠ‡—–ƒ‰‡‡•‹•’”‘–‡‹•—ǯƒ†
—’”‡˜‡–Ž‡–ŠƒŽˆ”ƒ‡•Š‹ˆ–•™Š‹Ž‡‹…”‡ƒ•‹‰„ƒ•‡•—„•–‹–—–‹‘—–ƒ–‹‘•Ǥ‘ŽǤ
‡ŽŽ2ǡͳͻͳǦͳͻͻǤ
ͳʹ
ƒ‰ǡǤǡ”—…ǡǤǡ”‹–ŒƒǡǤǡ—”‡”ǡǤǡ”ƒǡǤ
Ǥǡ‘‘†‰ƒ–‡ǡǤǡǯ‘‡ŽŽǡǤƒ†
‘‘†ƒǡ Ǥ Ǥ ȋͳͻͻͺȌ ‹‘…Š‡‹…ƒŽ „ƒ•‹• ‘ˆ Ǧ‹†—…‡† —–ƒ‰‡‡•‹• ‹
Escherichiacoliǣ”‡…‘•–‹–—–‹‘‘ˆinvitroŽ‡•‹‘„›’ƒ••†‡’‡†‡–‘–Š‡—ǯʹ
—–ƒ‰‡‹……‘’Ž‡šƒ†‡…’”‘–‡‹Ǥ”‘…Ǥƒ–ŽǤ…ƒ†Ǥ…‹Ǥ95ǡͻ͹ͷͷǦͻ͹͸ͲǤ
ͳ͵
…‡”›ǡ Ǥ Ǥ ƒ† ”‘™ǡ Ǥ ȋͳͻ͹ͻȌ ’‘Ž›‡”ƒ•‡ ‘ˆ Escherichia coliǤ
—”‹ˆ‹…ƒ–‹‘ƒ†‹†‡–‹ˆ‹…ƒ–‹‘‘ˆ•—„—‹–•ǤǤ‹‘ŽǤŠ‡Ǥ254ǡͳ͹ͶͺǦͳ͹ͷ͵Ǥ
ͳͶ
ƒ‹ǡ Ǥǡ ‘”‹—…Š‹ǡ Ǥ ƒ† ‘”„‡”‰ǡ Ǥ ȋͳͻͺͷȌ Š‡ ’‘Ž›‡”ƒ•‡ •—„—‹– ‘ˆ ’‘Ž›‡”ƒ•‡ ‘ˆ Escherichia coliǤ Ǥ ’Ž‹ˆ‹…ƒ–‹‘ ‘ˆ –Š‡ dnaE ‰‡‡ ’”‘†—…– ƒ†
’‘Ž›‡”ƒ•‡ƒ…–‹˜‹–›‘ˆ–Š‡Ƚ•—„—‹–ǤǤ‹‘ŽǤŠ‡Ǥ260ǡͳʹͻͺʹǦͳʹͻͺ͸Ǥ
ͳͷ
…Š‡—‡”ƒǡǤǤƒ†…Š‘Ž•ǡǤȋͳͻͺͶȌ•‡’ƒ”ƒ–‡‡†‹–‹‰‡š‘—…Ž‡ƒ•‡ˆ‘”
”‡’Ž‹…ƒ–‹‘ǣ–Š‡ɂ•—„—‹–‘ˆEscherichiacoli’‘Ž›‡”ƒ•‡Š‘Ž‘‡œ›‡Ǥ”‘…Ǥ
ƒ–ŽǤ…ƒ†Ǥ…‹Ǥ81ǡ͹͹Ͷ͹Ǧ͹͹ͷͳǤ
ͳ͸
ƒˆ–Ǧ‡œǡ Ǥ Ǥ ƒ† …Šƒƒ’‡” Ǥ Ǥ ȋʹͲͲͶȌ Š‡ Ʌ •—„—‹– ‘ˆ Escherichia coli ’‘Ž›‡”ƒ•‡ ǣ ƒ ”‘Ž‡ ‹ •–ƒ„‹Ž‹œ‹‰ –Š‡ ɂ ’”‘‘ˆ”‡ƒ†‹‰ •—„—‹–Ǥ Ǥ ƒ…–‡”‹‘ŽǤ 186ǡ
ʹ͹͹ͶǦʹ͹ͺͲǤ
ͳ͹
‘–‡ǡ Ǥǡ ‹…‡ŽŽ‹ǡ Ǥǡ …Šƒƒ’‡”ǡ Ǥ Ǥǡ ”‡••ƒ‹ǡ Ǥǡ –‡ˆƒǡ Ǥǡ ƒŽ ‹ƒœǡ Ǥ ƒ†
‘…Š‘‡’’Ž‡”ǡǤȋʹͲͳʹȌ–ƒ„‹Ž‹œƒ–‹‘‘ˆ–Š‡Escherichiacoli’‘Ž›‡”ƒ•‡ɂ
•—„—‹–„›–Š‡Ʌ•—„—‹–ˆƒ˜‘”•invivoƒ••‡„Ž›‘ˆ–Š‡‘Ž…ƒ–ƒŽ›–‹……‘”‡Ǥ”…ŠǤ
‹‘…Š‡Ǥ‹‘’Š›•Ǥ523ǡͳ͵ͷǦͳͶ͵Ǥ
ʹʹ
ͳͺ
”‹–…Šƒ”†ǡǤǤǡƒŽŽƒǡǤ
Ǥƒ†…‡”›ǡǤǤȋͳͻͻ͸Ȍ˜‹˜‘ƒ••‡„Ž›‘ˆ–Š‡ɒǦ
…‘’Ž‡š ‘ˆ –Š‡ ’‘Ž›‡”ƒ•‡ Š‘Ž‘‡œ›‡ ‡š’”‡••‡† ˆ”‘ ƒ ˆ‹˜‡Ǧ‰‡‡
ƒ”–‹ˆ‹…‹ƒŽ ‘’‡”‘Ǥ Ž‡ƒ˜ƒ‰‡ ‘ˆ –Š‡ ɒǦ…‘’Ž‡š –‘ ˆ‘” ƒ ‹š‡† ɀǦɒǦ…‘’Ž‡š „› –Š‡
’’”‘–‡ƒ•‡ǤǤ‹‘ŽǤŠ‡Ǥ271ǡͳͲʹͻͳǦͳͲʹͻͺǤ
ͳͻ
…‡”‡›ǡ Ǥǡ ‘Š•‘ǡ Ǥǡ ƒ–œǡ Ǥ ƒ† ǯ‘‡ŽŽǡ Ǥ ȋʹͲͲ͹Ȍ Šƒ”ƒ…–‡”‹œƒ–‹‘ ‘ˆ ƒ
–”‹’Ž‡’‘Ž›‡”ƒ•‡”‡’Ž‹•‘‡Ǥ‘ŽǤ‡ŽŽ27ǡͷʹ͹Ǧͷ͵ͺǤ
ʹͲ
—”‰‡”•ǡǤǤǡ‘”„‡”‰ǡǤƒ†ƒƒ‹„ƒ”ƒǡǤȋͳͻͺͳȌŠ‡dnaN‰‡‡…‘†‡•ˆ‘”–Š‡
Ⱦ •—„—‹– ‘ˆ ’‘Ž›‡”ƒ•‡ Š‘Ž‘‡œ›‡ ‘ˆ Escherichia coliǤ ”‘…Ǥ ƒ–ŽǤ …ƒ†Ǥ
…‹Ǥ78ǡͷ͵ͻͳǦͷ͵ͻͷǤ
ʹͳ
‘‰ǡ Ǥ Ǥǡ ”—•–ǡ Ǥǡ ǯ‘‡ŽŽǡ Ǥ ƒ† —”‹›ƒǡ Ǥ ȋͳͻͻʹȌ Š”‡‡Ǧ†‹‡•‹‘ƒŽ
•–”—…–—”‡‘ˆ–Š‡Ⱦ•—„—‹–‘ˆE.coli’‘Ž›‡”ƒ•‡Š‘Ž‘‡œ›‡ǣƒ•Ž‹†‹‰
…Žƒ’Ǥ‡ŽŽ69ǡͶʹͷǦͶ͵͹Ǥ
ʹʹ
‡†ƒǡ Ǥǡ …ƒ…‡ǡ Ǥ ƒ† ‘”„‡”‰ǡ Ǥ ȋͳͻ͹ͺȌ dnaB ’”‘–‡‹ ‘ˆ Escherichia coliǤ
—”‹ˆ‹…ƒ–‹‘ƒ†”‘Ž‡‹–Š‡”‡’Ž‹…ƒ–‹‘‘ˆ‫ͳכ‬͹ͶǤǤ‹‘ŽǤŠ‡Ǥ253ǡʹ͸ͳǦʹ͸ͻǤ
ʹ͵
‘™‡ǡǤƒ†‘”„‡”‰ǡǤȋͳͻ͹ͺȌ”‹ƒ•‡ǡ–Š‡dnaG’”‘–‡‹‘ˆEscherichiacoliǤ
‡œ›‡™Š‹…Š•–ƒ”–•…Šƒ‹•ǤǤ‹‘ŽǤŠ‡Ǥ253ǡ͹ͷͺǦ͹͸ͶǤ
ʹͶ
†‹ƒ‹ǡ Ǥǡ ƒ‰•–‘ǡ Ǥ Ǥǡ —”‹‡˜ƒǡ Ǥǡ ‘‘†ƒǡ Ǥ Ǥ ƒ† ǯ‘‡ŽŽǡ Ǥ ȋʹͲͲͻȌ
”ƒ•Ž‡•‹‘ ’‘Ž›‡”ƒ•‡• ”‡‘†‡Ž –Š‡ ”‡’Ž‹•‘‡ ƒ† ƒŽ–‡” –Š‡ •’‡‡† ‘ˆ –Š‡
”‡’Ž‹…ƒ–‹˜‡Š‡Ž‹…ƒ•‡Ǥ”‘…Ǥƒ–ŽǤ…ƒ†Ǥ…‹Ǥ106ǡ͸Ͳ͵ͳǦ͸Ͳ͵ͺǤ
ʹͷ
ƒ—ŽǡǤǤ†—––‘ǡǤǤȋʹͲͲͷȌ‘Ž‡•‘ˆ–Š‡Escherichiacoli‡…’”‘–‡‹ƒ†
–Š‡‰Ž‘„ƒŽ”‡•’‘•‡‹‡ˆˆ‡…–‹‰’‘Ž›‡”ƒ•‡•‡Ž‡…–‹‘‹˜‹˜‘ǤǤƒ…–‡”‹‘ŽǤ
187ǡ͹͸Ͳ͹Ǧ͹͸ͳͺǤ
ʹ͵
ʹ͸
ƒŽŽ‹ǡ Ǥǡ ‘’‘†‹ǡ Ǥ Ǥǡ ƒ•‘ǡ Ǥ Ǥ † ‘•–‡”ǡ Ǥ Ǥ ȋʹͲͳͷȌ –‡”ƒ…–‹‘• ƒ†
Ž‘…ƒŽ‹œƒ–‹‘‘ˆEscherichiacoli‡””‘”Ǧ’”‘‡’‘Ž›‡”ƒ•‡ƒˆ–‡”†ƒƒ‰‡ǤǤ
ƒ…–‡”‹‘ŽǤ197ǡʹ͹ͻʹǦʹͺͲͻǤ
ʹ͹
”—„‡”ǡǤǤǡ”†‡ǡǤǤǡƒ„ƒ–ǡ
Ǥǡƒ”ƒ–ƒǡǤǡƒ•œ…œ—”ǡǤǤǡ‘ǡǤǤǡŽ•‡ǡǤǤǡ
‘‘†‰ƒ–‡ǡ Ǥǡ ‘‘†ƒǡ Ǥ Ǥ ƒ† ‘šǡ Ǥ Ǥ ȋʹͲͳͷȌ ‡… ’”‘–‡‹ •—”ˆƒ…‡
”‡“—‹”‡†ˆ‘”ƒ…–‹˜ƒ–‹‘‘ˆ’‘Ž›‡”ƒ•‡Ǥ‘
‡‡–Ǥ11ǡ‡ͳͲͲͷͲ͸͸Ǥ
ʹͺ
†‹ƒ‹ǡǤǡƒ–‡ŽǡǤǡ
‘‘†ƒǡǤǤƒ†ǯ‘‡ŽŽǡǤȋʹͲͳ͵Ȍ‡…ƒ…–•ƒ•ƒ•™‹–…Š
–‘”‡‰—Žƒ–‡’‘Ž›‡”ƒ•‡‘……—’ƒ…›‹ƒ‘˜‹‰”‡’Ž‹…ƒ–‹‘ˆ‘”Ǥ”‘…Ǥƒ–ŽǤ…ƒ†Ǥ…‹Ǥ
110ǡͷͶͳͲǦͷͶͳͷǤ
ʹͻ
ƒǡ Ǥ Ǥǡ Šƒǡ Ǥ Ǥǡ —”—‘Š”‹ǡ Ǥǡ ƒ‹ǡ Ǥ ƒ† ‹›ƒƒǡ Ǥ Ǥ ȋʹͲͳͷȌ
‡…‘„‹ƒ•‡ ƒ† –”ƒ•Ž‡•‹‘ ’‘Ž›‡”ƒ•‡ †‡…”‡ƒ•‡ –Š‡ •’‡‡† ‘ˆ ”‡’Ž‹…ƒ–‹‘
ˆ‘”’”‘‰”‡••‹‘†—”‹‰–Š‡†ƒƒ‰‡”‡•’‘•‡‹EscherichiacolicellsǤ—…Ž‡‹…
…‹†•‡•Ǥ43ǡͳ͹ͳͶǦͳ͹ʹͷǤ
͵Ͳ
Žƒ†‡ǡ Ǥ ƒ† ƒ†ƒǡ Ǥ ȋʹͲͳͳȌ š‹†ƒ–‹˜‡ •–”‡•• ”‡•‹•–ƒ…‡ ‹ Deinococcus
radioduransǤ‹…”‘„‹‘ŽǤ‘ŽǤ‹‘ŽǤ‡˜Ǥ75ǡͳ͵͵ǦͳͻͳǤ
͵ͳ
†‡”•‘ǡǤǤǡ‘”†ƒǡǤǤǡƒ‹ǡǤǤǡƒ””‹•Šǡ
Ǥƒ†—‰‰ƒǡǤȋͳͻͷ͸Ȍ–—†‹‡•
‘ƒ”ƒ†‹‘Ǧ”‡•‹•–ƒ–‹…”‘…‘……—•ǤǤ•‘Žƒ–‹‘ǡ‘”’Š‘Ž‘‰›ǡ…—Ž–—”ƒŽ…Šƒ”ƒ…–‡”‹•–‹…•ǡ
ƒ†”‡•‹•–ƒ…‡–‘‰ƒƒ”ƒ†‹ƒ–‹‘Ǥ‘‘†‡…Š‘ŽǤ10ǡͷ͹ͷǦͷ͹͹Ǥ
͵ʹ
ƒ•‡ǡǤǤȋͳͻ͹ͺȌ—Ž–‹’Ž‹…‹–› ‘ˆ‰‡‘‡‡“—‹˜ƒŽ‡–•‹–Š‡”ƒ†‹ƒ–‹‘Ǧ”‡•‹•–ƒ–
„ƒ…–‡”‹—MicrococcusradioduransǤǤƒ…–‡”‹‘ŽǤ134ǡ͹ͳǦ͹ͷǤ
͵͵
ƒ”•‘Œ‘ǡ‹–ƒ›ƒƒǡǤƒ†ƒ–•—›ƒƒǡǤȋͳͻͺͳȌ
‡‘‡—Ž–‹’Ž‹…‹–›ƒ†”ƒ†‹ƒ–‹‘
”‡•‹•–ƒ…‡‹MicrococcusradioduransǤǤ‹‘…Š‡Ǥ90ǡͺ͹͹ǦͺͺͲǤ
͵Ͷ
Š‹–‡ǡ Ǥ ‡– ƒŽǤ ȋͳͻͻͻȌ ‡‘‡ •‡“—‡…‡ ‘ˆ –Š‡ ”ƒ†‹‘”‡•‹•–ƒ– „ƒ…–‡”‹—
DeinococcusradioduransͳǤ…‹‡…‡286ǡͳͷ͹ͳǦͳͷ͹͹Ǥ
ʹͶ
͵ͷ
—–ƒǡ Ǥ Ǥǡ —…Š•ǡ Ǥǡ —›ƒ‰ǡ Ǥ ƒ† ‹–‘ǡ Ǥ Ǥ ȋͳͻͻ͵Ȍ †‡–‹ˆ‹…ƒ–‹‘ǡ
•‡“—‡…‹‰ǡƒ†–ƒ”‰‡–‡†—–ƒ‰‡‡•‹•‘ˆƒ’‘Ž›‡”ƒ•‡‰‡‡”‡“—‹”‡†ˆ‘”–Š‡
‡š–”‡‡”ƒ†‹‘”‡•‹•–ƒ…‡‘ˆDeinococcusradioduransǤǤƒ…–‡”‹‘ŽǤ175ǡ͵ͷͺͳǦ͵ͷͻͲǤ
͵͸
Žƒ†‡ǡ Ǥǡ ‹†‡”ǡ Ǥ Ǥǡ ƒ—Žǡ Ǥ ƒ† ƒ†ƒǡ Ǥ ȋʹͲͲͻȌ ‡…‘„‹ƒ–‹‘ ƒ†
”‡’Ž‹…ƒ–‹‘ ‹ ”‡’ƒ‹” ‘ˆ Š‡ƒ˜‹Ž› ‹””ƒ†‹ƒ–‡† Deinococcus radioduransǤ ‡ŽŽ 136ǡ
ͳͲͶͶǦͳͲͷͷǤ
͵͹
‡…‘‹–‡ǡ Ǥǡ Š‡˜‡Ž‡˜ǡ Ǥ Ǥǡ ƒ‹Ž‘‡ǡ Ǥǡ ‘‡”ǡ Ǥ ƒ† ò„•…Š‡”ǡ Ǥ ȋʹͲͲͶȌ
˜‘Ž˜‡‡–‘ˆƒˆƒ‹Ž›’‘Ž›‡”ƒ•‡‹†‘—„Ž‡Ǧ•–”ƒ†‡†„”‡ƒ”‡’ƒ‹”‹–Š‡
”ƒ†‹‘”‡•‹•–ƒ–‘”‰ƒ‹•DeinococcusradioduransǤ‘ŽǤ‹…”‘„‹‘ŽǤ531ǡͳ͹ʹͳǦͳ͹͵ͲǤ
͵ͺ
‘•‡Ž‡›ǡǤǤǡƒ––‹‰Ž›ǡǤǡƒ†Š‹‹ǡǤȋͳͻ͹ʹȌ•‘Žƒ–‹‘ƒ†•‘‡’”‘’‡”–‹‡•
‘ˆ –‡’‡”ƒ–—”‡Ǧ•‡•‹–‹˜‡ —–ƒ–• ‘ˆ Micrococcus radiodurans †‡ˆ‡…–‹˜‡ ‹ •›–Š‡•‹•ǤǤ
‡Ǥ‹…”‘„‹‘ŽǤ70ǡ͵ͻͻǦͶͲͻǤ
͵ͻ
‡–…Š‹‘—ǡ Ǥǡ ‡”˜ƒ–ǡ Ǥǡ ‘•–‡ǡ Ǥ ƒ† ‘‡”ǡ Ǥ ȋʹͲͳͲȌ ƒŒ‘” ”‘Ž‡ ‘ˆ –Š‡
‡…’ƒ–Š™ƒ›‹†‘—„Ž‡Ǧ•–”ƒ†Ǧ„”‡ƒ”‡’ƒ‹”–Š”‘—‰Š‹Deinococcus
radioduransǤŽ‘
‡‡–Ǥ6ǡ‡ͳͲͲͲ͹͹ͶǤ
ͶͲ
‘•‡Ž‡›ǡǤǤǡƒ––‹‰Ž›ǡǤƒ†‘’Žƒ†ǡǤǤȋͳͻ͹ʹȌ‡•‹–‹œƒ–‹‘–‘”ƒ†‹ƒ–‹‘„›
Ž‘••‘ˆ”‡…‘„‹ƒ–‹‘ƒ„‹Ž‹–›‹ƒ–‡’‡”ƒ–—”‡Ǧ•‡•‹–‹˜‡—–ƒ–‘ˆMicrococcus
radioduransŠ‡Ž†ƒ–‹–•”‡•–”‹…–‹˜‡–‡’‡”ƒ–—”‡ǤǤ
‡Ǥ‹…”‘„‹‘ŽǤ72ǡ͵ʹͻǦ͵͵ͺǤ
Ͷͳ
ƒŽ›ǡǤǤǡ—›ƒ‰ǡǤǡ—…Š•ǡǤƒ†‹–‘ǡǤǤȋͳͻͻͶȌInvivo†ƒƒ‰‡ƒ†”‡…Ǧ
†‡’‡†‡– ”‡’ƒ‹” ‘ˆ ’Žƒ•‹† ƒ† …Š”‘‘•‘ƒŽ ‹ –Š‡ ”ƒ†‹ƒ–‹‘Ǧ”‡•‹•–ƒ–
„ƒ…–‡”‹—DeinococcusradioduransǤǤƒ…–‡”‹‘ŽǤ176ǡ͵ͷͲͺǦ͵ͷͳ͹Ǥ
Ͷʹ
‡‹œǡǤƒ†ƒ”šǡǤȋʹͲͲ͹Ȍ‡•‹‘„›’ƒ••ƒ…–‹˜‹–›‘ˆ’‘Ž›‡”ƒ•‡ˆ”‘–Š‡
‡š–”‡‡Ž› ”ƒ†‹‘”‡•‹•–ƒ– ‘”‰ƒ‹• Deinococcus radioduransǤ Ǥ ‹‘ŽǤ Š‡Ǥ 282ǡ
ͳͲͻͲͺǦͳͲͻͳͶǤ
ʹͷ
Ͷ͵
Žƒ•‹—•ǡ Ǥǡ Š‡˜‡Ž‡˜ǡ Ǥǡ ‘Ž‹˜‡–ǡ Ǥǡ ‘‡”ǡ Ǥ ƒ† ò„•…Š‡”ǡ Ǥ ȋʹͲͲͶȌ ’‘Ž›‡”ƒ•‡ ˆ”‘ Deinococcus radiodurans ’‘••‡••‡• ƒ •–”—…–—”‡Ǧ‘†—Žƒ–‡†
͵ǯÎͷǯ‡š‘—…Ž‡ƒ•‡ƒ…–‹˜‹–›‹˜‘Ž˜‡†‹”ƒ†‹‘”‡•‹•–ƒ…‡Ǥ‘ŽǤ‹…”‘„‹‘ŽǤ60ǡͳ͸ͷǦͳ͹͸Ǥ
ͶͶ
Šƒ‹”ƒ”ǡ Ǥ Ǥ ƒ† ‹•”ƒǡ Ǥ Ǥ ȋʹͲͲͻȌ ’‘Ž›‡”ƒ•‡ ˆ”‘ Deinococcus
radiodurans‹’Ž‹…ƒ–‡†‹„ƒ…–‡”‹ƒŽ–‘Ž‡”ƒ…‡–‘†ƒƒ‰‡‹•…Šƒ”ƒ…–‡”‹œ‡†ƒ•ƒ
•Š‘”–’ƒ–…Š„ƒ•‡‡š…‹•‹‘”‡’ƒ‹”’‘Ž›‡”ƒ•‡Ǥ‹…”‘„‹‘ŽǤ155ǡ͵ͲͲͷǦ͵ͲͳͶǤ
Ͷͷ
‡—ŽŽ‹‘–ǡ Ǥǡ Žƒ†‹°”‡ǡ Ǥǡ ‡…‘‹–‡ǡ Ǥǡ —”ƒ†ǡ Ǥǡ ò„•…Š‡”ǡ Ǥ ƒ† ‹Ž„‡—”‰Šǡ Ǥ
ȋʹͲͲͻȌŠ‡ˆƒ‹Ž›’‘Ž›‡”ƒ•‡ˆ”‘Deinococcusradioduransƒ†‘’–•ƒ‘Ǧ
•–ƒ†ƒ”†‡š–‡†‡†…‘ˆ‘”ƒ–‹‘ǤǤ‹‘ŽǤŠ‡Ǥ284ǡͳͳͻͻʹǦͳͳͻͻͻǤ
Ͷ͸
”‡••ƒ‹ǡǤǡ–‡ˆƒǡǤǡƒŽ‹ƒœǡǤǡ‹ƒ…Š‡––ƒǡǤǡ‡‰‰‹ƒ‹ǡǤƒ†‘…Š‘‡’’Ž‡”ǡǤ
ȋʹͲͲͻȌ”‘–‡‘Ž›•‹•‘ˆ–Š‡’”‘‘ˆ”‡ƒ†‹‰•—„—‹–…‘–”‘Ž•–Š‡ƒ••‡„Ž›‘ˆEscherichia
coli’‘Ž›‡”ƒ•‡…ƒ–ƒŽ›–‹……‘”‡Ǥ‹‘…Š‹Ǥ‹‘’Š›•Ǥ…–ƒ1794ǡͳ͸Ͳ͸Ǧͳ͸ͳͷǤ
Ͷ͹
‘–א ‹Ž˜ƒǡ Ǥǡ ƒ’‡–ƒǡ Ǥǡ –‡ˆƒǡ Ǥǡ ƒŽ ‹ƒœǡ Ǥǡ ‡……ƒ”‡ŽŽ‹ǡ Ǥǡ ‡””‘‡ǡ Ǥ ƒ†
‘…Š‘‡’’Ž‡”ǡ Ǥ ȋʹͲͳͷȌ ‹—Ž–ƒ‡‘—• –‡”ƒ”› ‡š–‡•‹‘ ‘ˆ …ƒ–ƒŽ›œ‡† „› ƒ
–”‹‡”‹…”‡’Ž‹…ƒ•‡ƒ••‡„Ž‡†invivoǤ‹‘…Š‡Ǥ‹‘’Š›•Ǥ‡•Ǥ‘Ǥ462ǡͳͶǦʹͲǤ
Ͷͺ
—‹ŽŽ± —ž”‡œǡ Ǥ Ǥǡ –‡ˆƒǡ Ǥǡ ‡ƒǡ Ǥǡ ‘–‡ǡ Ǥ ƒ† ‘…Š‘‡’’Ž‡”ǡ Ǥ ȋʹͲͳʹȌ
‘–‹—‘—• ‡œ›‡Ǧ…‘—’Ž‡† ƒ••ƒ› ‘ˆ ’Š‘•’Šƒ–‡Ǧ ‘” ’›”‘’Š‘•’Šƒ–‡Ǧ”‡Ž‡ƒ•‹‰
‡œ›‡•Ǥ‹‘‡…Š‹“—‡•53ǡͻͻǦͳͲ͵Ǥ
Ͷͻ
…Š‡‹„‡ǡǤǡ‡”–ǡǤƒ†‡”‰‡›‡”ǡǤǤȋͳͻ͹ͶȌ”‹…ƒ…‹†ǣǦƒ••ƒ›™‹–Š—”‹…ƒ•‡ǡ
’Ǥ ͳͻͻͻǦʹͲͲͷǤ ǣ Ǥ Ǥ ‡”‰‡›‡” ȋ†ǤȌǡ ‡–Š‘†‡ †‡” œ›ƒ–‹•…Š‡ ƒŽ›•‡ǡ
‡”Žƒ‰Š‡‹‡ǡ‡‹Š‡‹ȋȌǤ
ͷͲ
…Ž—”‡ǡ Ǥ Ǥ ȋͳͻ͸ͻȌ‹‡–‹…ƒƒŽ›•‹• ‘ˆ …‘—’Ž‡† ‡œ›‡ ƒ••ƒ›•Ǥ ‹‘…Š‡‹•–”› 8ǡ
ʹ͹ͺʹǦʹ͹ͺ͸Ǥ
ʹ͸
ͷͳ
”ƒ†ˆ‘”†ǡ Ǥ Ǥ ȋͳͻ͹͸Ȍ ”ƒ’‹† ƒ† •‡•‹–‹˜‡ ‡–Š‘† ˆ‘” –Š‡ “—ƒ–‹–ƒ–‹‘ ‘ˆ
‹…”‘‰”ƒ “—ƒ–‹–‹–‡• ‘ˆ ’”‘–‡‹ —–‹Ž‹œ‹‰ –Š‡ ’”‹…‹’Ž‡ ‘ˆ ’”‘–‡‹Ǧ†›‡ „‹†‹‰Ǥ
ƒŽǤ‹‘…Š‡Ǥ72ǡʹͶͺǦʹͷͶǤ
ͷʹ
”‘™ǡ Ǥ Ǥǡ —…ƒǡ Ǥ Ǥ ƒ† ƒ’„‡ŽŽǡ Ǥ Ǥ ȋͳͻͻ͵Ȍ —”‹ˆ‹…ƒ–‹‘ ƒ†
…Šƒ”ƒ…–‡”‹œƒ–‹‘‘ˆ–Š‡Saccharomycescerevisiae’‘Ž›‡”ƒ•‡Ɂ‘˜‡”’”‘†—…‡†
‹EscherichiacoliǤǤ‹‘ŽǤŠ‡Ǥ268ǡͻͺʹǦͻͻͲǤ
ͷ͵
ƒŽ›ǡ Ǥ Ǥǡ ƒ‹†ƒƒ‘˜ƒǡ Ǥ Ǥǡ ƒ–”‘•‘˜ƒǡ Ǥ Ǥǡ ƒ•‹Ž‡‘ǡ Ǥǡ Šƒ‹ǡ Ǥǡ
‡ƒ–‡•™ƒ”ƒǡǤǡ‡••ǡǤǡ‡Ž…Š‡‘ǡǤǤǡ‘•–ƒ†ƒ”‹–Š‡•ǡǤǤǡƒƒ”‘˜ƒǡǤ
Ǥǡƒ…‡––ǡǤǤǡ”‡†”‹…•‘ǡǤǤƒ†
Š‘•ƒŽǡǤȋʹͲͲͶȌ……——Žƒ–‹‘‘ˆȋȌ‹
Deinococcusradioduransˆƒ…‹Ž‹–ƒ–‡•‰ƒƒǦ”ƒ†‹ƒ–‹‘”‡•‹•–ƒ…‡Ǥ…‹‡…‡306ǡͳͲʹͷǦ
ͳͲʹͺǤ
ͷͶ
”ƒǡǤ
Ǥƒ†‘‘†‰ƒ–‡ǡǤȋʹͲͲ͹Ȍ…”‡ƒ•‡†…ƒ–ƒŽ›–‹…ƒ…–‹˜‹–›ƒ†ƒŽ–‡”‡†ˆ‹†‡Ž‹–›
‘ˆ Š—ƒ ’‘Ž›‡”ƒ•‡ ɇ ‹ –Š‡ ’”‡•‡…‡ ‘ˆ ƒ‰ƒ‡•‡Ǥ Ǥ ‹‘ŽǤ Š‡Ǥ 282ǡ
ʹͶ͸ͺͻǦʹͶ͸ͻ͸Ǥ
ͷͷ
‘”Œƒǡ Ǥ Ǥǡ ‡œƒ ‘”ƒǡ Ǥǡ ƒ””אǡ Ǥǡ ‘••‡–ǡ Ǥǡ ƒÀ”‡œǡ Ǥ Ǥ ƒ† ƒ”ƒǡ Ǥ Ǥ
ȋʹͲͳʹȌ ‰‹‡‡”‹‰ Escherichia coli –‘ ‹…”‡ƒ•‡ ’Žƒ•‹† ’”‘†—…–‹‘ ‹ Š‹‰Š
…‡ŽŽǦ†‡•‹–›…—Ž–‹˜ƒ–‹‘•‹„ƒ–…Š‘†‡Ǥ‹…”‘„Ǥ‡ŽŽƒ…–Ǥ11ǡͳ͵ʹǤ
ͷ͸
…Š—––‡ǡǤǤƒ†‘šǡǤǤȋͳͻͺ͹Ȍ‘‘Ž‘‰›Ǧ†‡’‡†‡–…Šƒ‰‡•‹ƒ†‡‘•‹‡ͷǯǦ
–”‹’Š‘•’Šƒ–‡ Š›†”‘Ž›•‹• †—”‹‰ recA ’”‘–‡‹ ’”‘‘–‡† •–”ƒ† ‡š…Šƒ‰‡ǣ ‡˜‹†‡…‡
ˆ‘”Ž‘‰’ƒ”ƒ‡‹……‘’Ž‡š‡•Ǥ‹‘…Š‡‹•–”›26ǡͷ͸ͳ͸Ǧͷ͸ʹͷǤ
ͷ͹
—ǡǤƒ†‰‡ŽƒǡǤǤȋͳͻͻʹȌ–”—…–—”ƒŽ†ƒ–ƒ•—‰‰‡•––Šƒ––Š‡ƒ…–‹˜‡ƒ†‹ƒ…–‹˜‡
ˆ‘”• ‘ˆ –Š‡ ‡… ˆ‹Žƒ‡– ƒ”‡ ‘– •‹’Ž› ‹–‡”…‘˜‡”–‹„Ž‡Ǥ Ǥ ‘ŽǤ ‹‘ŽǤ 227ǡ ͵͵ͶǦ
͵Ͷ͸Ǥ
ʹ͹
ͷͺ
‡†ƒŽ‡ǡǤǤƒ†‘šǡǤǤȋͳͻͻ͸Ȍ˜‹†‡…‡ˆ‘”–Š‡…‘—’Ž‹‰‘ˆŠ›†”‘Ž›•‹•–‘
–Š‡ˆ‹ƒŽȋ‡š–‡•‹‘Ȍ’Šƒ•‡‘ˆ‡…’”‘–‡‹Ǧ‡†‹ƒ–‡†•–”ƒ†‡š…Šƒ‰‡ǤǤ‹‘ŽǤ
Š‡Ǥ271ǡͷ͹ʹͷǦͷ͹͵ʹǤ
ͷͻ
—ǡ Ǥǡ ƒ…‘„•ǡ Ǥ Ǥǡ ‡•–ǡ Ǥ Ǥǡ ‰ƒ™ƒǡ Ǥ ƒ† ‰‡Žƒǡ Ǥ Ǥ ȋʹͲͲͳȌ ‘ƒ‹
•–”—…–—”‡ƒ††›ƒ‹…•‹–Š‡Š‡Ž‹…ƒŽˆ‹Žƒ‡–•ˆ‘”‡†„›‡…ƒ†ƒ†ͷͳ‘Ǥ
”‘…Ǥƒ–ŽǤ…ƒ†Ǥ…‹Ǥ98ǡͺͶͳͻǦͺͶʹͶǤ
͸Ͳ
ƒ‘‘…ǡǤǤǡ—ǡǤǡƒ‰ǡǤǡƒ‹ǡǤǤǡ‘™ǡǤǡƒ’„‡ŽŽǡǤǤƒ†‰‡ŽƒǡǤǤ
ȋʹͲͲ͵Ȍ Ǧ‡†‹ƒ–‡† …‘ˆ‘”ƒ–‹‘ƒŽ …Šƒ‰‡• ‹ –Š‡ ‡… ˆ‹Žƒ‡–Ǥ –”—…–—”‡ 11ǡ
ͳͺ͹Ǧͳͻ͸Ǥ
͸ͳ
ƒ”—–ƒǡ Ǥǡ —ǡ Ǥǡ ƒ‰ǡ Ǥǡ ‰‡Žƒǡ Ǥ Ǥ ƒ† ‘šǡ Ǥ Ǥ ȋʹͲͲ͵Ȍ ’ƒ‹”‹‰Ǧ
‡Šƒ…‡† …‘ˆ‘”ƒ–‹‘ ‘ˆ „ƒ…–‡”‹ƒŽ ‡… ’”‘–‡‹•Ǥ Ǥ ‹‘ŽǤ Š‡Ǥ 278ǡ ͷʹ͹ͳͲǦ
ͷʹ͹ʹ͵Ǥ
͸ʹ
—•‡––‹ǡǤǤǡŠƒ™ǡǤǤƒ†‘šǡǤǤȋʹͲͲ͵Ȍƒ‰‡•‹—‹‘Ǧ†‡’‡†‡–ƒ…–‹˜ƒ–‹‘
‘ˆ–Š‡‡…’”‘–‡‹‹˜‘Ž˜‡•–Š‡–‡”‹—•ǤǤ‹‘ŽǤŠ‡Ǥ278ǡͳ͸͵ͺͳǦͳ͸͵ͺͺǤ
͸͵
‹ǡǤǡŠƒ”ƒǡǤǤǡ„„‘––ǡǤǤǡ‘‘†ǡǤǤǡ™›‡”ǡǤǤǡƒ„—”ƒǡǤǡ‹–‘ǡǤ
ǤǡƒǡǤǤǡƒŽ›ǡǤǤƒ†‘šǡǤǤȋʹͲͲʹȌ‡…’”‘–‡‹ˆ”‘–Š‡‡š–”‡‡Ž›
”ƒ†‹‘”‡•‹•–ƒ– „ƒ…–‡”‹— Deinococcus radioduransǣ ‡š’”‡••‹‘ǡ ’—”‹ˆ‹…ƒ–‹‘ǡ ƒ†
…Šƒ”ƒ…–‡”‹œƒ–‹‘ǤǤƒ…–‡”‹‘ŽǤ184ǡͳ͸ͶͻǦͳ͸͸ͲǤ
͸Ͷ
‹ǡ Ǥ ƒ† ‘šǡ Ǥ Ǥ ȋʹͲͲʹȌ Š‡ ‡… ’”‘–‡‹• ‘ˆ Deinococcus radiodurans ƒ†
Escherichia coli ’”‘‘–‡ •–”ƒ† ‡š…Šƒ‰‡ ˜‹ƒ ‹˜‡”•‡ ’ƒ–Š™ƒ›•Ǥ ”‘…Ǥ ƒ–ŽǤ
…ƒ†Ǥ…‹Ǥ99ǡ͹ͻͳ͹Ǧ͹ͻʹͳǤ
ʹͺ
FIGURELEGENDS
Figure1
ƒ”–‘‘ƒ†•‡“—‡…‡•‘ˆ–Š‡†‹ˆˆ‡”‡–•—•‡†‹–Š‡’”‡•‡–™‘”Ǥ
Figure2
’‘Ž›‡”ƒ•‡ƒ…–‹˜‹–‹‡•†‡–‡…–‡†™‹–ŠȽ”ƒ†Ƚ…ƒ––Š‡‡š’‡•‡‘ˆ†‹ˆˆ‡”‡–•ǤŽŽ
–Š‡ƒ…–‹˜‹–›˜ƒŽ—‡•ƒ”‡‡š’”‡••‡†ƒ•–Š‡‹…”‡ƒ•‡‘ˆ’›”‘’Š‘•’Šƒ–‡…‘…‡–”ƒ–‹‘ȋȌ’‡”
—‹––‹‡ȋ•ȌǤȋAȌŽ‘‰ƒ–‹‘”ƒ–‡‘ˆͶͲ‡”†•„›͵ͶȽ”—†‡”…‘†‹–‹‘•‘ˆŽ‘™
‹‘‹…•–”‡‰–Šȋ‘–”‘Žǡ†ƒ””‡†„ƒ”Ȍǡ‘”‹–Š‡’”‡•‡…‡‘ˆʹͲͲƒŽȋ„Ž—‡„ƒ”Ȍ‘”Ž
ȋ’‹„ƒ”ȌǤȋBȌš–‡•‹‘”ƒ–‡‘ˆͶͲ‡”†•„›͵͵Ƚ…—†‡”…‘†‹–‹‘•‘ˆŽ‘™‹‘‹…
•–”‡‰–Šȋ‘–”‘Žǡ†ƒ””‡†„ƒ”Ȍǡ‘”‹–Š‡’”‡•‡…‡‘ˆʹͲͲƒŽȋ„Ž—‡„ƒ”ȌǤȋCȌƒ–‡‘ˆ
ƒ…–‹˜ƒ–‡†…ƒŽˆ–Š›—•‡Ž‘‰ƒ–‹‘…ƒ–ƒŽ›œ‡†„›͵ͶȽ”ȋ‰”‡‡„ƒ”Ȍ‘”„›͵͵Ƚ…
ȋ‘”ƒ‰‡„ƒ”ȌǤ
Figure3
ƒ•‡ƒ…–‹˜‹–›‘ˆEscherichiacoli‡…ƒ†ƒ••ƒ›•‘ˆȽ…‡š–‡•‹‘ƒ…–‹˜‹–›ǤȋAȌŠ‡
”‡Ž‡ƒ•‡‘ˆ‘”–Š‘’Š‘•’Šƒ–‡ˆ”‘…ƒ–ƒŽ›œ‡†„›ͳɊ‡…™ƒ•ƒ••ƒ›‡†‹–Š‡’”‡•‡…‡
‘ˆͶͲ‡”••ȋ‰”‡‡†‘–•Ȍ‘”ƒ––Š‡‡š’‡•‡‘ˆͶͲ‡”†•ȋ„Ž—‡†‘–•ȌǤȋBȌ••ƒ›‘ˆ
–Š‡ ‘”–Š‘’Š‘•’Šƒ–‡ ”‡Ž‡ƒ•‡† „› ͳ Ɋ ‡… ˆ”‘ ͳ  ƒ† ‘ˆ –Š‡ ’›”‘’Š‘•’Šƒ–‡
‰‡‡”ƒ–‡†„›͵͵Ƚ…‹–Š‡’”‡•‡…‡‘ˆͶͺ‡”†•ƒ†͸Ͳ‡”••ǡͳɊ‡ƒ…Š
ȋ•‡‡ ‹‰—”‡ ͳȌǤ ‡ƒ…–‹‘ ‹š–—”‡• …‘–ƒ‹‡† ‹‘•‹‡ǡ ƒ† ƒ•‡ǡ ƒ•‡ǡ ƒ† ƒ•
…‘—’Ž‹‰‡œ›‡•Ǥ‡ƒ…–‹‘•™‡”‡•–ƒ”–‡†™‹–Š†
ƒ††ȋͳͲͲɊ‡ƒ…Šǡ‰”‡‡†‘–•Ȍǡ
†
ȋ„Ž—‡†‘–•Ȍǡ‘”†ȋ”‡††‘–•ȌǤȋCȌ••ƒ›‘ˆ–Š‡’›”‘’Š‘•’Šƒ–‡‰‡‡”ƒ–‡†„›͵͵
ʹͻ
Ƚ…‹–Š‡’”‡•‡…‡‘ˆͶͺ‡”†•ƒ†͸Ͳ‡”••ǡͳɊ‡ƒ…Šǡ‹–Š‡ƒ„•‡…‡‘ˆ‡…Ǥ
–Š‡”…‘†‹–‹‘•ƒ•‹‹‰—”‡͵Ǥ
Figure4
••ƒ›•‘ˆEscherichiacoli‡…ƒ•‡ƒ…–‹˜‹–›ƒ†‘ˆȽ…”‡…‘„‹ƒ–‹‘Ǧ†‡’‡†‡–
‡Ž‘‰ƒ–‹‘Ǥ ȋAȌ ‹ˆˆ‡”‡…‡ ƒ„•‘”’–‹‘ •’‡…–”ƒ ”‡…‘”†‡† ͷǡ ͳͷǡ ʹͷǡ ƒ† ͵ͷ ‹—–‡• ȋ’‹ǡ
”‡†ǡ„Ž—‡ǡƒ†‰”‡‡Ž‹‡•ǡ”‡•’‡…–‹˜‡Ž›Ȍƒˆ–‡”ƒ††‹–‹‘‘ˆͳɊ‡…–‘ƒ”‡ƒ…–‹‘‹š–—”‡
…‘–ƒ‹‹‰ ͳ  ǡ ͳ Ɋ ͸Ͳ‡” ••ǡ ‹‘•‹‡ǡ ǡ ȾǦǡ ƒ•‡ǡ ǡ ǡ ƒ†
Ǥ …—˜‡––‡ …‘–ƒ‹‹‰ ƒ ”‡ƒ…–‹‘ ‹š–—”‡ †‡˜‘‹† ‘ˆ ‡… ™ƒ• —•‡† ƒ• ”‡ˆ‡”‡…‡Ǥ ȋBȌ
„•‘”„ƒ…‡ †‹ˆˆ‡”‡…‡• ƒ• ƒ ˆ—…–‹‘ ‘ˆ –‹‡ †‡–‡…–‡† ƒ– ʹͻ͵ ȋ†‹ƒ‘†•Ȍ ƒ† ͵ͶͲ 
ȋ…‹”…Ž‡•Ȍ —†‡” –Š‡ …‘†‹–‹‘• ‘ˆ ‹‰—”‡ ͳǤ ȋCȌ ‹ˆˆ‡”‡…‡ ƒ„•‘”’–‹‘ •’‡…–”ƒ ”‡…‘”†‡†
†—”‹‰–Š‡ˆ‹”•–ͳͲ‹—–‡•ƒˆ–‡”ƒ††‹–‹‘‘ˆ͵͵Ƚ…–‘ƒ”‡ƒ…–‹‘‹š–—”‡…‘–ƒ‹‹‰ͳ
Ɋ‡…ǡͳǡͶͺ‡”†•ƒ†͸Ͳ‡”••ȋͳɊ‡ƒ…ŠȌǡͳͲͲɊ†ǡ‹‘•‹‡ǡ
ǡȾǦǡƒ•‡ǡƒ•‡ǡǡǡƒ†Ǥ…—˜‡––‡…‘–ƒ‹‹‰ƒ”‡ƒ…–‹‘‹š–—”‡
†‡˜‘‹† ‘ˆ ‡… ƒ† Ƚ… ™ƒ• —•‡† ƒ• ”‡ˆ‡”‡…‡Ǥ ȋDȌ ‹‡–‹…• ‘ˆ ’›”‘’Š‘•’Šƒ–‡ ”‡Ž‡ƒ•‡
‡š‡”–‡† „› Ƚ… †—”‹‰ ”‡…‘„‹ƒ–‹‘Ǧ†‡’‡†‡– ‡Ž‘‰ƒ–‹‘ ȋ†‹ƒ‘†•Ȍǡ ƒ† –‹‡
…‘—”•‡ ‘ˆ ‡… ƒ•‡ ƒ…–‹˜‹–› ȋ…‹”…Ž‡•Ȍǡ —†‡” –Š‡ …‘†‹–‹‘• ‘ˆ ‹‰—”‡ ͶǤ Š‡ ‡…
ƒ•‡ƒ…–‹˜‹–›™ƒ•†‡–‡”‹‡†„›“—ƒ–‹–ƒ–‹‰–Š‡…‘—’Ž‡†‘š‹†ƒ–‹‘‘ˆȾǦǡƒ†–Š‡
”‡Ž‡ƒ•‡‘ˆ’›”‘’Š‘•’Šƒ–‡„›Ƚ…™ƒ•…ƒŽ…—Žƒ–‡†˜‹ƒ–Š‡…‘—’Ž‡†‰‡‡”ƒ–‹‘‘ˆ—”‹…ƒ…‹†ǡ–‘
–Š‡–‘–ƒŽ…‘…‡–”ƒ–‹‘‘ˆ™Š‹…Š™ƒ••—„–”ƒ…–‡†–Š‡…‘…‡–”ƒ–‹‘‰‡‡”ƒ–‡†„›–Š‡ƒ…–‹‘
‘ˆ‡…Ǥ
͵Ͳ
Figure5
‡…‘„‹ƒ–‹‘Ǧ†‡’‡†‡–‡š–‡•‹‘ƒ…–‹˜‹–›‘ˆȽ”ƒ†Ƚ…ƒ••ƒ›‡†‹–Š‡’”‡•‡…‡
‘ˆ‡…ǤȋAȌ‹ˆˆ‡”‡…‡ƒ„•‘”’–‹‘•’‡…–”ƒ”‡…‘”†‡†‡˜‡”›ͳͲ‹—–‡•ƒˆ–‡”ƒ††‹–‹‘‘ˆ͵Ͷ
Ƚ”–‘ƒ”‡ƒ…–‹‘‹š–—”‡…‘–ƒ‹‹‰ͳɊ‡…ǡͳǡͶͺ‡”†•ƒ†͸Ͳ‡”
•• ȋͳ Ɋ ‡ƒ…ŠȌǡ ͳͲͲ Ɋ †ǡ ‹‘•‹‡ǡ ǡ ȾǦǡ ƒ•‡ǡ ƒ•‡ǡ ǡ ǡ ƒ†
Ǥ …—˜‡––‡ …‘–ƒ‹‹‰ ƒ ”‡ƒ…–‹‘ ‹š–—”‡ †‡˜‘‹† ‘ˆ ‡… ƒ† Ƚ” ™ƒ• —•‡† ƒ•
”‡ˆ‡”‡…‡Ǥ ȋBȌ ‹‡–‹…• ‘ˆ –‘–ƒŽ ‘”–Š‘’Š‘•’Šƒ–‡ ”‡Ž‡ƒ•‡† „› –Š‡ ƒ…–‹‘ ‘ˆ ‡… ƒ† Ƚ”
ȋˆ‹ŽŽ‡†…‹”…Ž‡•Ȍǡ‘ˆ’›”‘’Š‘•’Šƒ–‡‰‡‡”ƒ–‡†„›Ƚ”ȋ†‹ƒ‘†•Ȍǡƒ†‘ˆȾǦ‘š‹†ƒ–‹‘
Ž‹‡†–‘‡…ƒ…–‹‘ȋ‡’–›…‹”…Ž‡•ȌǤȋCȌ‡–ƒ‹Ž‘ˆ–Š‡‹‡–‹…•”‡’‘”–‡†‹‹‰—”‡ͷǡƒ†
”‡’”‡•‡–‹‰ –Š‡ ’›”‘’Š‘•’Šƒ–‡ ‰‡‡”ƒ–‡† „› –Š‡ ”‡…‘„‹ƒ–‹‘Ǧ†‡’‡†‡– ‡š–‡•‹‘ƒ…–‹˜‹–›‘ˆȽ”ǤȋDǦFȌ‡…‘„‹ƒ–‹‘Ǧ†‡’‡†‡–’‘Ž›‡”ƒ•‡ƒ…–‹˜‹–›‘ˆ͵͵
Ƚ…ǡƒ•†‡–‡”‹‡†—†‡”–Š‡•ƒ‡…‘†‹–‹‘•‘ˆ‹‰—”‡•ͷǦǤ
Figure6
‡…‘„‹ƒ•‡ƒ…–‹‘‘ˆ‡…ƒ†”‡…‘„‹ƒ–‹‘Ǧ†‡’‡†‡–‡š–‡•‹‘ƒ…–‹˜‹–›‘ˆȽ”
ƒ†Ƚ…ƒ••ƒ›‡†‹–Š‡’”‡•‡…‡‘”‹–Š‡ƒ„•‡…‡‘ˆ‡…ǤŠ‡ƒ…–‹˜‹–›˜ƒŽ—‡•‘ˆ‡…ƒ”‡
‡š’”‡••‡† ƒ• –Š‡ ‹…”‡ƒ•‡ ‘ˆ ’Š‘•’Šƒ–‡ …‘…‡–”ƒ–‹‘ ȋȌǡ ‰‡‡”ƒ–‡† ƒ– –Š‡ ‡š’‡•‡ ‘ˆ
ǡ’‡”—‹––‹‡ȋ•ȌǤŠ‡‡š–‡•‹‘ƒ…–‹˜‹–›˜ƒŽ—‡•ƒ”‡‡š’”‡••‡†ƒ•–Š‡‹…”‡ƒ•‡‘ˆ
’›”‘’Š‘•’Šƒ–‡…‘…‡–”ƒ–‹‘ȋȌ’‡”—‹––‹‡ȋ•ȌǤȋAȌƒ•‡ƒ…–‹˜‹–›‘ˆ‡…†‡–‡…–‡†
‹ –Š‡ ’”‡•‡…‡ ‘ˆ Ƚ” ‘” Ƚ… ȋ‰”‡‡ ƒ† „Ž—‡ „ƒ”ǡ ”‡•’‡…–‹˜‡Ž›ȌǤ Š‡ ’‘Ž›‡”ƒ•‡
ƒ…–‹˜‹–› ‘ˆ Ƚ” ƒ† Ƚ… ƒ”‡ ”‡’‘”–‡† ™‹–Š †ƒ” ‰”‡‡ ƒ† †ƒ” „Ž—‡ „ƒ”•ǡ ”‡•’‡…–‹˜‡Ž›Ǥ
••ƒ›…‘†‹–‹‘•ƒ•‹‹‰—”‡ͷǤȋBȌ‡…‘„‹ƒ–‹‘Ǧ†‡’‡†‡–‡š–‡•‹‘ƒ…–‹˜‹–›‘ˆ
Ƚ” ȋ‰”‡‡ „ƒ”Ȍ ƒ† Ƚ… ȋ„Ž—‡ „ƒ”Ȍ ƒ••ƒ›‡† ‹ –Š‡ ƒ„•‡…‡ ‘ˆ ‡…Ǥ Š‡ …‘””‡•’‘†‹‰
‹‡–‹…•ƒ”‡”‡’‘”–‡†‹—’’Ž‡‡–ƒ”›‹‰—”‡ͶǤ
͵ͳ
Figure7
‡…‘„‹ƒ–‹‘Ǧ†‡’‡†‡–‡š–‡•‹‘ƒ…–‹˜‹–›‘ˆȽ”ȋʹͷɊ‰‘ˆȀȌ‘˜‡”‡š’”‡••‡†
‹ •‘Ž—„Ž‡ ˆ‘” ƒ••ƒ›‡† ‹ –Š‡ ’”‡•‡…‡ ‘ˆ ‡…Ǥ ȋAȌ ‹ˆˆ‡”‡…‡ ƒ„•‘”’–‹‘ •’‡…–”ƒ
”‡…‘”†‡† ‡˜‡”› ͳͲ ‹—–‡• ƒˆ–‡” ƒ††‹–‹‘ ‘ˆ Ƚ” –‘ ƒ ”‡ƒ…–‹‘ ‹š–—”‡ …‘–ƒ‹‹‰ ͳ Ɋ
‡…ǡ ͳ  ǡ Ͷͺ‡” †• ƒ† ͸Ͳ‡” •• ȋͳ Ɋ ‡ƒ…ŠȌǡ ͳͲͲ Ɋ †ǡ ‹‘•‹‡ǡ
ǡȾǦǡƒ•‡ǡƒ•‡ǡǡǡƒ†Ǥ…—˜‡––‡…‘–ƒ‹‹‰ƒ”‡ƒ…–‹‘‹š–—”‡
†‡˜‘‹† ‘ˆ ‡… ƒ† Ƚ” ™ƒ• —•‡† ƒ• ”‡ˆ‡”‡…‡Ǥ ȋBȌ ‹‡–‹…• ‘ˆ –‘–ƒŽ ‘”–Š‘’Š‘•’Šƒ–‡
”‡Ž‡ƒ•‡†„›–Š‡ƒ…–‹‘‘ˆ‡…ƒ†Ƚ”ȋˆ‹ŽŽ‡†…‹”…Ž‡•Ȍǡ‘ˆ’›”‘’Š‘•’Šƒ–‡‰‡‡”ƒ–‡†„›Ƚ”
ȋ†‹ƒ‘†•Ȍǡ ƒ† ‘ˆ ȾǦ ‘š‹†ƒ–‹‘ Ž‹‡† –‘ ‡… ƒ…–‹‘ȋ‡’–› …‹”…Ž‡•ȌǤ ȋCȌ ‡–ƒ‹Ž ‘ˆ
–Š‡‹‡–‹…•”‡’‘”–‡†‹‹‰—”‡͹ǡƒ†”‡’”‡•‡–‹‰–Š‡’›”‘’Š‘•’Šƒ–‡‰‡‡”ƒ–‡†„›–Š‡
”‡…‘„‹ƒ–‹‘Ǧ†‡’‡†‡– ‡š–‡•‹‘ ƒ…–‹˜‹–› ‘ˆ Ƚ” ȋˆ‹ŽŽ‡† †‹ƒ‘†•ȌǤ • ƒ
…‘’ƒ”‹•‘ǡ –Š‡ ”‡…‘„‹ƒ–‹‘Ǧ†‡’‡†‡– ’‘Ž›‡”ƒ•‡ ƒ…–‹˜‹–› ‘ˆ ͵Ͷ  ”‡ˆ‘Ž†‡†
Ƚ”‹•ƒŽ•‘”‡’‘”–‡†ȋ‡’–›†‹ƒ‘†•ȌǤ
Figure8
‹‡–‹…•‘ˆ‡…Ǧ†‡’‡†‡–‡š–‡•‹‘…ƒ–ƒŽ›œ‡†„›Ƚ”ƒ†Ƚ…ǤȋAȌ‡–ƒ‹Ž‘ˆ‹‰—”‡
ͷ •Š‘™‹‰ –Š‡ ‡š–‡•‹‘ ‘ˆ ȋ†‹ƒ‘†•Ȍ ƒ† –Š‡ Š›†”‘Ž›•‹• ‘ˆ ȋˆ‹ŽŽ‡† …‹”…Ž‡•Ȍ
…ƒ–ƒŽ›œ‡† „› Ƚ” ƒ† ‡…ǡ ”‡•’‡…–‹˜‡Ž›ǡ †—”‹‰ –Š‡ ˆ‹”•– ͺͲ ‹—–‡• ƒˆ–‡” ”‡ƒ…–‹‘•
•–ƒ”–‡†Ǥ ȋBȌ ‡–ƒ‹Ž ‘ˆ ‹‰—”‡ ͷ •Š‘™‹‰ –Š‡ ‡š–‡•‹‘ ‘ˆ ȋ†‹ƒ‘†•Ȍ ƒ† –Š‡
Š›†”‘Ž›•‹•‘ˆȋˆ‹ŽŽ‡†…‹”…Ž‡•Ȍ…ƒ–ƒŽ›œ‡†„›Ƚ…ƒ†‡…ǡ”‡•’‡…–‹˜‡Ž›ǡ†—”‹‰–Š‡ˆ‹”•–
ͺͲ ‹—–‡• ƒˆ–‡” ”‡ƒ…–‹‘• •–ƒ”–‡†Ǥ Š‡ …‘…‡–”ƒ–‹‘ ‘ˆ ’›”‘’Š‘•’Šƒ–‡ ƒ• ƒ ˆ—…–‹‘ ‘ˆ
–‹‡ ȋ…‘””‡•’‘†‹‰ –‘ ‘Ž›‡”ƒ•‡ ƒ…–‹˜‹–›Ȍ ™ƒ• …ƒŽ…—Žƒ–‡† •—„–”ƒ…–‹‰ –‘ –Š‡
‘„•‡”˜‡†–‘–ƒŽ‘”–Š‘’Š‘•’Šƒ–‡…‘…‡–”ƒ–‹‘–Š‡ˆ”ƒ…–‹‘‰‡‡”ƒ–‡†„›‡…ǡƒ††‹˜‹†‹‰
„›ʹ–Š‡”‡•‹†—ƒŽ•Ǥ
͵ʹ
Scheme1
‡ƒ…–‹‘’ƒ–Š•Ž‡ƒ†‹‰–‘–Š‡‰‡‡”ƒ–‹‘‘ˆ—”‹…ƒ…‹†ƒ––Š‡‡š’‡•‡‘ˆ‘”–Š‘’Š‘•’Šƒ–‡ƒ†
‹‘•‹‡ȋ—’’‡””‘™Ȍǡƒ†–‘–Š‡‘š‹†ƒ–‹‘‘ˆȾǦˆ”‘ƒ†’Š‘•’Š‘‡‘Ž’›”—˜ƒ–‡
ȋŽ‘™‡” ”‘™ȌǤ Š‹Ž‡ –Š‡ †‡–‡”‹ƒ–‹‘ ‘ˆ —”‹… ƒ…‹† …ƒ „‡ —•‡† –‘ ƒ••ƒ› „‘–Š ‘Ž›‡”ƒ•‡ƒ†‡…ƒ…–‹˜‹–›ǡ–Š‡‡˜ƒŽ—ƒ–‹‘‘ˆȾǦ‘š‹†ƒ–‹‘‹•—•‡ˆ—Ž–‘•’‡…‹ˆ‹…ƒŽŽ›
ƒ••ƒ›‡…‹–Š‡’”‡•‡…‡‘ˆ…‘…‘‹–ƒ–‘Ž›‡”ƒ•‡ƒ…–‹˜‹–›Ǥ
Table1
†‡–‹ˆ‹…ƒ–‹‘ „› ƒ•• •’‡…–”‘•…‘’› ‘ˆ ’ƒ”–‹ƒŽŽ› ’—”‹ˆ‹‡† Deinococcus radiodurans ƒ
‘Ž›‡”ƒ•‡Ǥ
Table2
†‡–‹ˆ‹…ƒ–‹‘ „› ƒ•• •’‡…–”‘•…‘’› ‘ˆ Escherichia coli ‘Ž›‡”ƒ•‡ Ⱦ •—„—‹–ǡ –Š‡
’”‡•‡…‡ ‘ˆ ™Š‹…Š ™ƒ• †‡–‡…–‡† ƒ• …‘–ƒ‹ƒ– ‘ˆ Deinococcus radiodurans ƒ
‘Ž›‡”ƒ•‡ǡ‘˜‡”‡š’”‡••‡†‹•‘Ž—„Ž‡ˆ‘”ƒ†•—„Œ‡…–‡†–‘ˆ‘—”…‘•‡…—–‹˜‡’—”‹ˆ‹…ƒ–‹‘
•–‡’•Ǥ
Table3
†‡–‹ˆ‹…ƒ–‹‘ „› ƒ•• •’‡…–”‘•…‘’› ‘ˆ Escherichia coli ‘Ž›‡”ƒ•‡ Ƚ •—„—‹–ǡ –Š‡
’”‡•‡…‡ ‘ˆ ™Š‹…Š ™ƒ• †‡–‡…–‡† ƒ• …‘–ƒ‹ƒ– ‘ˆ Deinococcus radiodurans ƒ
‘Ž›‡”ƒ•‡ǡ‘˜‡”‡š’”‡••‡†‹•‘Ž—„Ž‡ˆ‘”ƒ†•—„Œ‡…–‡†–‘ˆ‘—”…‘•‡…—–‹˜‡’—”‹ˆ‹…ƒ–‹‘
•–‡’•Ǥ
͵͵
7DEOH3HSWLGHVRID. radiodurans'1$3RO\PHUDVH,,,ĮVXEXQLWLGHQWLILHGE\PDVVVSHFWURPHWU\
3HSWLGH
2EVHUYHG0U'D 7KHRUHWLFDO0U'D
6HTXHQFH
6'$7$+(7//$,47.$
6/$(/*($'$$5
,71/'3/()(//)(5
((/71/93905
69(',*/,.
6*$)'$)*(5
66,$3<6'/(5
($/*/<,6*+3/(4+(*/5
7DEOH3HSWLGHVRIE. coli51$3RO\PHUDVHȕVXEXQLWLGHQWLILHGE\PDVVVSHFWURPHWU\
3HSWLGH
2EVHUYHG0U'D
7KHRUHWLFDO0U'D
7DEOH3HSWLGHVRIE. coli51$3RO\PHUDVHĮVXEXQLWLGHQWLILHGE\PDVVVSHFWURPHWU\
3HSWLGH
2EVHUYHG0U'D
7KHRUHWLFDO0U'D