Genetics 301
Lecture 13 -Telomeres
Cooper review
The end replication problem
-during lagging strand replication a primase creates RNA/DNA
hybrids to which DNA polymerase α binds and extends to form
okazaki fragments
-RNA primers removed and replaced with DNA by DNA
polymerase δ
-at 5’ end, DNA pol δ can’t bind to fill in 3’ overhang (see d in fig.
below)
-for every round of replication, the DNA would shorten
-problem solved by a terminal 3’ overhang which allows priming
outside of “useful” DNA – this overhang is part of the Telomere
-telomeres also prevent fusion of chromosome ends through blunt
ends (i.e., they cap chromosomes)
-telomere allows full synthesis of DNA end as it is now primed
from overhang (see f below)
Telomerase
-However, now leading strand synthesis can’t create terminal 3’
overhang during replication of opposite strand
-this overhang is recreated by telomerase
-telomerase uses a built in RNA primer to extend the 3’ end –
it’s a reverse transcriptase
-the DNA needs to be chewed back a bit in the 5’-3’ direction
to produce a 3’ overhang complementary to the telomerase
RNA
- in metazoans, most somatic cells do not express telomerase –
therefore, losing telomere sequence with every cell division
-at a certain point of loss, the cells become senescent and can no
longer replicate their DNA and divide
-certain cancers express telomerase and can then re-enter the cell
cycle
-Telomere position effect
-silencing proteins (Sir2-4) bind to telomeres and sub-telomeric
regions
-Sir2 is and ADP-ribosylase and deacetylase – activity leads to
nucleosome tightening and heterochromatin formation
-when genes are placed near telomeres, they become
transcritpionally inactive because of heterochromatin
-How is telomere length maintained?
-a set of proteins (Rap1, Rif1/2, Ku) that bind to double stranded
telomeric DNA provide a negative feedback signal when they
reach a threshold level on the telomere
– the existing telomere determines the length of the newly
synthesized telomere
-if lagging strand polymerases are defective, the existing
telomere remains single stranded for some time
-the synthesis of new telomere on leading strand continues
beyond normal time because there is no signal from lagging
strand to stop – telomeres become unusually long
-Capping of telomeres
-some of the proteins at telomere also prevent fusion of
chromosome ends
-this role of telomeres is independent of the requirement for
maintaining chromosome length
-in telomerase and Rap1 mutants chromosome fusion occurs even
before telomere becomes critically shortened
-DNA damage repair
-telomeres also serve as a reservoir for proteins (Ku, Sir3/4)
involved in non-homologous end joining (NHEJ), a process that
repairs double stranded DNA breaks
-proteins leave telomeres when breaks occur and go to site of break
-TPE is reduced when cells have double-stranded breaks
(DSBs) due to reduction of Sir proteins at subtelomeric DNA
-these proteins might help to stabilize DNA aroung breaks to help
repair
-there is a paradox that these proteins aid repair of DSBs by NHEJ
while at same time help to prevent NHEJ of telomeres when at the
telomeres – there must be something about telomere sequence or
other proteins there that alter the function of Ku and Sir proteins
-Meitotic chromosome segregation and recombination
-during meiosis telomeres associate with centrosome
-thought to help in sorting of homologous chromosomes, and
facilitate recombination between homologs
- in Rap1 mutants, telomeres do not bind to centrosome and
cells are defective for recombination and segregation
-in fission yeast, nucleus moves around rapidly pulled by
centrosome and draggin chromosomes behind – helps
recombination between homologs
-in dynein mutants, nucleus doesn’t move around but
homologs still attach to centrosome - recombination is
defective (nuclear movement required) but homologous
chromosomes are properly segregated (telomere
attachment is sufficient)
-Cell cycle regulation of telomeres
-telomerase activity is altered during cell cycle
-not much known about signals
-mutants in DNA damage checkpoint signaling proteins show
telomere defects, some as severe as telomerase mutant, but not
known how they interact with telomere
-ADP ribosylases important for genomic stability (eg. PARP, Sir2)
are associated with telomeres
-sumoylation (like ubiquitoylation, but doesn’t lead to protein
degradation) might regulate telomere association with nuclear pore
complexes