Bi/Ch 111 - Biochemistry of Gene Expression
Winter Quarter 2017
Professor Judith Campbell
and
Professor Carl Parker
Tuesdays and Thursdays, 11:00-11:55 AM - 147 Noyes
Recitation: As arranged
California Institute of Technology
Bi/Ch 111 - Biochemistry of Gene Expression
Winter Quarter 2017
Professors Campbell and Parker
LECTURE SCHEDULE-CAMPBELL
Topic
Lecture 1 - General Features of DNA Replication (Feb. 7, 2017)
Lecture 2 - Enzymology of DNA Replication: DNA Polymerase I of E.
coli
Lecture 3 - Enzymology of DNA Replication: Replicative DNA
Polymerases Helicases
Lecture 4 - Enzymology of DNA Replication: Topoisomerases
Lecture 5 - Initiation of DNA Replication –Prof. William Dunphy
Lecture 6 – In vitro Replication-Biochemical methods of studying DNA
replication
Lecture 7 – Mismatch Repair
Lecture 8 - DNA Repair and Mutation Avoidance
Lecture 9 - RNA interference (small RNA-mediated pathways that
regulate gene expression) Prof. Alexei Aravin
Lecture 10 - DNA Recombination
California Institute of Technology
Bi/Ch 111 - Biochemistry of Gene Expression
Winter Quarter 2017
Professors Campbell and Parker
Assigned Reading
in Molecular Biology by Weaver
Lecture
1
General Features of DNA Replication - Chapter 20.1, pp. 637-646; Chapter
21.3, 702-716
2-3
DNA Polymerase I of E. coli and Replicative DNA Polymerases; Helicases Chapter 20.2, pp. 646-656; Chapter 21.2, pp. 694-705
4
Chapter 20.2, pp. 650-664
5-7
Chapter 20.1, 20.2 and Chapter 21
8
Chapter 20.3
9
Chapter 16.6-16.9
10
Chapter 22, 23
WEDNESDAY RECITATIONS 2017
Dates
Feb. 15
Lagging strand synthesis and coupling of leading and lagging strands
Feb. 22
Ribonucleotides in DNA
March 1
Reconstitution of eukaryotic DNA replication
March 8
Review for exam
Recitation 1:
Background paper:
Jin YH, Ayyagari R, Resnick MA, Gordenin DA, Burgers PM (2003). Okazaki fragment
maturation in yeast. II. Cooperation between the polymerase and 3'-5'-exonuclease
activities of Pol delta in the creation of a ligatable nick. J Biol Chem. 278(3):1626-33.
Assigned papers for presentation and discussion:
Garg, P., Stith, C. M., Sabouri, N., Johansson, E., and Burgers, P. M. (2004). Idling by
DNA polymerase {delta} maintains a ligatable nick during lagging-strand DNA
replication. Genes Dev 18, 2674-2673.
Hamdan et al., (2007) Dynamic DNA Helicase-DNA polymerase interactions assure
processive replication fork movement. Mol. Cell 27, 539-549.
Recitation 1:
For Background: Annu Rev Biochem. 2009;78:205-43.
Motors, switches, and contacts in the replisome.
Hamdan SM, Richardson CC.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School,
Boston, MA 02115, USA. [email protected]
Replisomes are the protein assemblies that replicate DNA. They function as molecular motors to
catalyze template-mediated polymerization of nucleotides, unwinding of DNA, the synthesis of
RNA primers, and the assembly of proteins on DNA. The replisome of bacteriophage T7
contains a minimum of proteins, thus facilitating its study. This review describes the molecular
motors and coordination of their activities, with emphasis on the T7 replisome. Nucleotide
selection, movement of the polymerase, binding of the processivity factor, unwinding of DNA,
and RNA primer synthesis all require conformational changes and protein contacts. Laggingstrand synthesis is mediated via a replication loop whose formation and resolution is dictated by
switches to yield Okazaki fragments of discrete size. Both strands are synthesized at identical
rates, controlled by a molecular brake that halts leading-strand synthesis during primer synthesis.
The helicase serves as a reservoir for polymerases that can initiate DNA synthesis at the
replication fork. We comment on the differences in other systems where applicable.
Especially notice the supplementary Material link for an animation of the models derived
from single molecule analysis: Nature. 2009 Jan 15;457(7227):336-9
Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis.
Hamdan SM, Loparo JJ, Takahashi M, Richardson CC, van Oijen AM.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School,
240 Longwood Avenue, Boston, Massachusetts 02115, USA.
In all organisms, the protein machinery responsible for the replication of DNA, the replisome, is
faced with a directionality problem. The antiparallel nature of duplex DNA permits the leadingstrand polymerase to advance in a continuous fashion, but forces the lagging-strand polymerase
to synthesize in the opposite direction. By extending RNA primers, the lagging-strand
polymerase restarts at short intervals and produces Okazaki fragments. At least in prokaryotic
systems, this directionality problem is solved by the formation of a loop in the lagging strand of
the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel
with the leading-strand polymerase. The replication loop grows and shrinks during each cycle of
Okazaki fragment synthesis. Here we use single-molecule techniques to visualize, in real time,
the formation and release of replication loops by individual replisomes of bacteriophage T7
supporting coordinated DNA replication. Analysis of the distributions of loop sizes and lag times
between loops reveals that initiation of primer synthesis and the completion of an Okazaki
fragment each serve as a trigger for loop release. The presence of two triggers may represent a
fail-safe mechanism ensuring the timely reset of the replisome after the synthesis of every
Okazaki fragment.
Annu. Rev. Biochem. 2009.78:205-243. Downloaded from arjournals.annualreviews.org
by California Institute of Technology on 01/06/10. For personal use only.
ANRV378-BI78-09
ARI
7 May 2009
14:58
primer synthesis in halting helicase movement.
In E. coli, the physical association of DnaG primase with DnaB helicase is sufficient, at least
in the absence of SSB protein, to cause a cessation of leading-strand DNA synthesis (78).
Both leading- and lagging-strand synthesis have
been reconstituted in the T7 replication system.
Under conditions in which synthesis is coordinated, multiple cycles of replication loop formation and release are observed (156a). This
real-time observation of the replication loop allows the construction of the time line of events
that leads to loop formation and release (see
below).
Timing Mechanism
of Replication Loop
Okazaki
fragment
Figure 8
Two models have been proposed for the release of the replication loop during each cycle of Okazaki fragment synthesis. Two movie
clips depict these two models in cartoon format. Follow the Supplemetary Material link
from the Annual Reviews home page at http://
www.annualreviews.org. In the collision
model (Figure 8), the encounter of the laggingstrand DNA polymerase with the 5′ -terminus
of the previously synthesized Okazaki fragment
triggers dissociation of the polymerase and subsequent loop release. In support of this model,
T4 DNA polymerase and E. coli DNA Pol III
holoenzyme dissociate when they encounter a
5′ -terminus while extending a primer on ssDNA (63, 230–232). The second model, the
signaling model (Figure 9), proposes that the
synthesis of a primer signals the release of
the replication loop even if the nascent Okazaki
fragment is not yet completed. Several lines
of evidence support this model. Gel and electron microscopic analysis show the existence
of gaps of ssDNA between Okazaki fragments
produced by the T4 replisome, indicating the
release of replication loops containing nascent
Okazaki fragments (233, 234). Furthermore,
the size of Okazaki fragments produced by the
E. coli, T4, and T7 replisomes is influenced
by the activity of the primase (221, 228, 233).
Varying the concentration of ATP and CTP
Collision release of the replication loop. In this frame from an animation by
Steve Moskowitz at Advanced Medical Graphics, the lagging-strand polymerase
is in the process of releasing the completed Okazaki fragment as a result of its
collision with the 5′ -end of the previously synthesized Okazaki fragment.
results in changes in the size of Okazaki fragments (221, 228, 233). Okazaki fragments size
is sensitive to primase-helicase interactions in
E. coli (235) and dependent on the concentration
of two important components for the laggingstrand synthesis by the T4 replisome, the clamp
and clamp loader (233).
To understand the molecular mechanisms
underlying replication loop dynamics, coordinated leading and lagging synthesis in the
T7 system has been observed at the singlemolecule level (156a). These studies show that
leading- and lagging-strand synthesis proceed
at identical rates during Okazaki fragment synthesis. They also reveal a lag time separating
replication loops, indicating that intermediate
steps are necessary for the formation of a new
replication loop. These studies demonstrate
that both the collision and signaling mechanisms occur in the T7 replication system and
thus account for the maintenance of a relatively
constant size of Okazaki fragments. In the signaling mechanism, the condensation of the first
nucleotide to yield pppAC results in release of
the loop. The subsequent extension of the dinucleotide to the functional tetranucleotide takes
www.annualreviews.org • Motors, Switches, and Contacts in the Replisome
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