Nucleic Acids: Manipulation, Structure and function

1
Nucleic Acids Manipulation, Structure and
function

• R. D. Gietz
• Associate Professor
• Department of Biochemistry and Medical Genetics.
• Tel. [phone]
• Email. gietz_at_cc.umanitoba.ca

2
Topics

• DNA Replication
• Initiation of DNA replication
• Replication of leading and lagging strands
• Telomere structure

3
Topics

• DNA Repair
• Base excision Repair
• Nucleotide Excision Repair
• Mismatch Repair

4
Topics

• In Vitro Mutagenesis
• Methods to make specific changes in DNA sequence.

5
Eukaryotic DNA Replication

• Highly regulated process.
• Cells Job replicate its DA exactly once pre
  cell cycle during S phase.
• Questions about control of DNA replication in
  eukaryotes
• How is DNA replication intitiated?
• How is it restricted to S phase?
• How can it occur only once per cell cycle?

6
How is DNA replication regulated?
Model proposed by F. Jacob and S. Brenner in
1964 for the regulation of DNA replication.
Thought to apply to both prokaryotes and
eukaryotes alike.
7
Cell fusion experiments showed there was control
over nuclear replication of DNA.

• P. N. Rao and R. T. Johnson, Nature225, 159
  (1970)
• F. Cross, J. Roberts, H. Weintraub, Annu. Rev.
  Cell Biol. 5, 341 (1989)
• See Figure 1

8
Figure 1
9
Conclusions

• Only chromosomes from G1 are competent to
  initiate DNA replication.
• S phase cells but not G1 or G2 contain an
  activator of replication
• G2 cells do not re-replicate until they pass thru
  mitosis.

10
Questions

• What is the nature of the replicator or ORI?
• What is causing the competent state for
  replication?
• What is the nature of the activator?
• What prevents re-replication?

11
Cell cycle control of replication ensures genome
integrity.
A is the normal cell cycle. B and C Checkpoints
ensure that the temporal order of cell cycle. C-1
block to re-replication in S phase. C-2 block to
re-replication in G2.
12
Control of re-replication is performed by Cyclin
dependent Kinases (CDKs)

• CDKs act as master controllers of cell cycle
  regulation.
• In S. cerevisiae (ScCdc28) and S. pombe (SpCdc2)
  contain a single CDK that controls cell cycle
  regulation.
• In mammalian cells Cdk2, 4 6 function at the
  onset of S phase.
• Cdk1(cdc2) operates in M phase.

13
CDKs control re-replication

• DNA replication is controlled sequentially by
  CDKs coupled with S phase and mitotic cyclins
  during the cell cycle.
• M phase CDK is dominant as fusion with M phase
  cells force S and G1 phase cells into M.
• What happens if the M phase CDK is inactivated in
  G2 in a cell?

14
CDK Controllers

• Temp sensitive mutations of SpCdc2 in activated
  in G2 cause the cells to re-replicate their DNA
  without going thru mitosis.
• cdc13 mutants become polyploid.
• What is cdc13? Sp mitotic cyclin.
• Re-replication also occurs when rum1(Sp) or
  SIC1(Sc) (CDK inhibitor) is ectopically expressed
• This shows that the mitotic CDK is also involved
  in preventing re-replication in addition to entry
  into Mitosis. See C1 C2

15
CDKs and Cyclins in Mammalin cell cycle
How is the transition accomplished, to assure
cell cycle moves forward? three broad methods
1) Phosphorylation/dephos of CDK (as described)
2)Specific inhibitors to regulate CDK activity.
3) Destruction of cyclin and inhibitors at
appropriate time in cell cycle
16
Cell cycle control of replication ensures genome
integrity.
A is the normal cell cycle. B and C Checkpoints
ensure that the temporal order of cell cycle. C-1
block to re-replication in S phase. C-2 block to
re-replication in G2.
17
How does one identify Origins? Or replicators
18
Cis acting replication elements

• To understand regulation of replication one must
  understand the replication origin.
• In eukaryotes the location of the origin of DNA
  replication is determined by cis-acting DNA
  sequences (origin of replication ORI).
• trans-acting proteins binds to the replicator.
• Eukaryotic chromosomes are too large to replicate
  from a single origin and so must contain multiple
  origins.

19
Cis Acting Elements

• In S. cerevisiae, replicators consist of multiple
  functional DNA elements, only one of which is
  essential (A element). H. Rao, Y. Marahrens, B.
  Stillman, Mol. Cell. Biol. 14, 7643 (1994)
• Next to the essential A element are two or three
  functionally conserved DNA elements (B1, B2, and
  B3) not individually essential but are necessary
  for initiation and influence the frequency with
  which an origin is used. H. Rao, Y. Marahrens, B.
  Stillman, Mol. Cell. Biol. 14, 7643 (1994)
• The A, B1, and B2 elements form the core of the
  replicator and bind essential DNA replication
  proteins, whereas the B3 element functions as a
  replicator enhancer by binding a protein called
  autonomously replicating sequence (ARS)-binding
  Factor 1 Abf1p. J. F. X. Diffley and B.
  Stillman, Proc. Natl. Acad. Sci. U.S.A. 85, 2120
  (1988)

20
Yeast Origin of replication
21
Trans acting Factors

• A multi-subunit protein called the origin
  recognition complex ORC that binds to the A and
  B1 elements in S. cerevisiae replicators was
  discovered in 1992 . P. Bell and B. Stillman,
  Nature 357, 128 (1992).
• ORC contains six polypeptides that are all
  essential for cell division and for the
  initiation of DNA replication. J. J. Li and I.
  Herskowitz, Science 262, 1870 (1993).
• ORC serves as a landing pad for protein-protein
  interactions that are regulated during the cell
  cycle and is used for controlling activation.

22
Yeast Origin Licensing

• In Yeast It has been shown that the ORC complex
  is bound to the ARS throughout the cell cycle.
  Not so for the mammalian equivalent.
• How then is the replication controlled?
• Through a process call ORIGIN licensing.

23
Origin Licensing

• Origin licensing is a stepwise process.
• In yeast the ORC complex is bound to the ori
  thoughout the cell cycle (this is not the case
  with mammalian cells).
• At the completion of mitotis the low CDK activity
  leads to the production of the pre-RC or licensed
  origin.
• High CDK activity during the rest of the cell
  cycle inhibit the formation of the pre- or
  licensed origin. See next figure

24
CDKs and Cyclins in Mammalin cell cycle
How is the transition accomplished, to assure
cell cycle moves forward? three broad methods
1) Phosphorylation/dephos of CDK (as described)
2)Specific inhibitors to regulate CDK activity.
3) Destruction of cyclin and inhibitors at
appropriate time in cell cycle
25
ORC complex in yeast

• Purified by identifying proteins that bound the
  ARS sequence.
• Orc 1 to 6 bind to the ARS sequence
• The binding requires ATP. ORC1 and 5 have ATP
  binding sites.
• All orc genes are required for DNA replication
  (deletion studies).
• ORC proteins have been identified in mammalian
  cells. However S. cerevisiae is the only ORC that
  binds to a specific sequence.

26
Origin licensing proteins

• Cdc6/18 is a protein that appears at the end of
  mitosis and disappears after the initiation of
  DNA replication. Depletion of cdc18 from S.pombe
  causes cells to not initiate DNA replication but
  go thru mitosis.
• Xenopus cells depleted for Cdc6/18 do not
  initiate DNA replication.

27
Origin licensing proteins

• Cdt1 (Cdc10 dependent transcript 1) is essential
  for initiation of replication. The protein peaks
  at G1/S and disappear after DNA replication has
  initiated. Cdt1 is found in human cells and
  behaves identically.
• Interesting ScCdt1 is produced in late G1 and
  excluded from the nucleus for the rest of the
  cell cycle.

28
Origin licensing proteins

• MCM proteins were identified in a yeast (Sc)
  screen that identified mutants that were
  defective in minichromosome maintenance.
• Family of 6 genes all related to each other and
  contain ATP binding motif.
• Form a Hexameric complex around 600 kDa.
• Work as a replicative helicase. Biochemical
  studies show helicase activity. Work as trimer
  complexes of Mcm2,3,5 (regulatory) and Mcm 4,6,7
  (catalytic).
• These proteins have also been found in Human
  cells.

29
Origin licensing
As a cell complete mitosis the ORC complex is
joined by the binding of Cdc6/18 and Cdt1 on the
Chromatin Cdc6/18 and Cdt1 function to
promote the binding of the Mcm complex. The
ori Is now considered to be licensed.
30
Activation of Licensed Origins

• CDKs and DDK protein kinase have a role in origin
  activation.
• CDKs associated with Sphase cyclins are involved.
  Cdc2-Cig2 Sp Cdc28-Clb5 or 6 Sc Cdk2-CyclinE
  in eukaryotes. Targets are still not defined.
• DDK (Dbf-4 dependent kinase) made up of Cdc7
  kinase subunit and regulatory Dbf4 subunit (this
  subunit if produced at G1-S transition).
• Activation by the Protein kinases lead to changes
  in the pre-RC and binding of Cdc45 and Mcm10 to
  the Mcm complex and unwinding of the DNA.
• DNA replication proteins such as RPA, DNA
  polymerases ? ? are then recruited to the ORI.

31
Targets of CDKs and DDK

• CDK targets. Sld2/Drc1 was recently shown to be a
  target
• Phosphorylated Sld2/Drc1 binds to ScDpb11 and the
  formation of this complex is essential for the
  following association of DNA polymerases on the
  replication origin. Masumoto, H., Muramatsu, S.,
  Kamimura, Y. Araki, H. (2002)Nature
  415,651655.
• Reports suggest that MCM proteins are likely
  targets of DDKs. Masai, H., Matsui, E., You, Z.,
  Ishimi, Y., Tamai, K. Arai, K. (2000) J. Biol.
  Chem. 275, 2904229052.
• Mcm2 was shown to be phosphorylated by Cdc7-Dbf4.
  Mcm2 protein is believed to inhibit the helicase
  activity of the MCM-4-6-7 complex, Mcm2
  phosphorylation by DDKs could cause a structural
  change in the MCM complex that would increase its
  affinity for Cdc45 and its helicase activity.
• DDKs may act locally at the replication origin,
  CDKs may function globally to initiate S-phase.
  (Pasero, P., Duncker, B.P., Schwob, E. Gasser,
  S.M. (1999). Genes Dev.13, 21592176.)
• DDKs could do so by phosphorylating MCM proteins
  at each origin while CDKs may be generally
  promoting initiation steps, including the
  formation of a Sld2/Drc1-Dbp11 complex.

32
Blocks to re-replication of DNA

• After initiation of DNA replication both the
  Cdc45 and the mcm complex move from the origin
  with the replication complex. This converts the
  ORI to an unlicensed state.
• Phosphorylation of Cdc6/18 by CDKs targets it for
  ubiquitin dependent degradation through the SCF
  complex. Cell cycle specific proteolysis,
  together with cell cycle specific transcription,
  ensures that the protein only accumulates in G1
  when the licensing is legitimate.
• Cdt1 protein levels are carefully controlled
  during the cell cycle to ensure that licensing
  only takes place during G1.

33
Mammalian Blocks to re-replication

• HsCdt1 is only present in G1 of the cell cycle
• Hs Cdc6/18 is present throughout the cell cycle
  and is only degraded for a short period at the
  end of M.
• ORC complex is not associate with chromatin
  throughout the cell cycle as in Sc. Orc1
  dissociates from chromatin and is degraded in S
  phase.
• Geminin is a component of APC-ubiquitin
  degradation system and binds to Ctd1. It is
  degraded at the end of Mitosis and releases Ctd1
  allowing licensing.

34
ORI Licensing and cell cycle
35
Figure legend

• DNA replication licensing control during the cell
  cycle.
• The cell cycle is separated into two
  stages, a period with no or low CDK activity in
  G1 (represented by light green in the cycle) and
  a period with increased CDK activity from the
  onset of S-phase to the end of M-phase
  (represented by light pink). After the
  completion of mitosis, DNA is licensed for
  replication by loading of the MCM complex on to
  chromatin. This process is only allowed when CDK
  activity is at very low levels, normally at the
  end of M-phase. When the cells are committed to
  a new cell cycle, CDKs and a second protein
  kinase, DDK, are activated, leading to the
  initiation of DNA replication. DNA replication
  enzymes are recruited and the MCM complex,
  probably acting as a replicative helicase, moves
  on the chromatin as elongation proceeds, together
  with the replication machinery. At the same
  time, the origin is converted to an unlicensed
  state. CDK dependent phosphorylation of licensing
  factors prevents re-licensing by inhibiting their
  chromatin binding or by targeting them for
  proteolysis or nuclear export. In metazoa,
  Geminin, present from the onset of S phase to the
  end of M phase, binds to Cdt1 and prevents
  licensing. When DNA duplication and chromosome
  segregation have been faithfully completed, CDKs
  are inactivated and Geminin is degraded.
  Completion of mitosis allows the
  dephosphorylation of proteins and accumulation of
  loading factors, thereby permitting a new round
  of licensing.

36
Mammalian ORI sequences?

• Despite the best efforts of researchers specific
  sequences can not be identified from mammalian
  cells that act like yeast origins.
• Much research has gone into in vitro replications
  systems in X. laevis which just seem to replicate
  any DNA presented to the system. Mahbubani, H.
  M., Paull, T., Elder, J. K. Blow, J. J. DNA
  replication initiates at multiple sites on
  plasmid DNA in Xenopus egg extracts. Nucleic
  Acids Res. 20, 14571462 (1992).
• This represented one group of researchers that
  thought there was no concensus DNA sequence that
  acted as the mammalian ORI.
• Specific regions of mammalian chromosomes have
  been identified as replicator sites or ORIs.

37
Eukaryotic Origins

• Both the human ? globin (HBB) and the DHFR locus,
  deletions 2550 Kb away from the region of
  replication-initiation activity also completely
  eliminated its activity.
• the HBB locus has a deletions (from ? thalassemia
  patients Lepore Hb) which remove sequences near
  the promoter for the ? globin gene and eliminate
  the origin located within the-globin locus. M. I.
  Aladjem et al., Science270, 815 (1995)
• This suggests that quite diverse DNA sequence
  elements can control the positioning of origins
  of DNA replication to specific sites within
  chromosomes, even elements that affect large
  chromosomal domains.

38
Eukaryotic Origins

• Recent studies moving these two regions to an
  ectopic site using viral recombinase show that
  they do have ORI actitivy.
• Wang, L. et al. The human ?-globin replication
  initiation region consists of two modular
  independent replicators. Mol. Cell. Biol. 24,
  33733386 (2004).
• Altman, A. L. Fanning, E. Defined sequence
  modules and an architectural element cooperate to
  promote initiation at an ectopic mammalian
  chromosomal replication origin. Mol. Cell. Biol.
  24, 41384150 (2004).

39
How do you identify a mammalian Origin?
Wang, L. et al. Mol. Cell. Biol. 24, 33733386
(2004).
Fig 1(A) Schematic illustration of the
methodology used for nascent-strand abundance
assay. DNA strands are depicted as solid gray
lines, and 5 primer RNAs are shown as solid black
boxes. Newly replicated origin-proximal DNA was
selected by size (600 to 2,500 bp) and by
resistance to lambda exonuclease (an enzyme that
digests DNA with a 5 DNA tail but not DNA with a
5 RNA tail). Nascent strands isolated in this way
were then subjected to real-time PCR with primers
encompassing the locus of interest.
40
How do you identify a mammalian Origin?
Wang, L. et al. Mol. Cell. Biol. 24, 33733386
(2004).
Fig 1(B) An example of real-time PCR output.
Standards of genomic DNA at fixed concentrations
were used in a PCR along with an unknown sample.
The abundance of the PCR products in nascent DNA
was calculated based on the cycle in which
fluorescence from the real-time PCR crossed the
manually set threshold. (C) A calibration curve
based on the data shown in panel B.
41
Beta globin replicator
Wang, L. et al. Mol. Cell. Biol. 24, 33733386
(2004).
Fig 1(F) A histogram depicting the average
abundance of specific sequences in nascent
strands, represented as the average of
the measurements shown in panel E from three
independent nascent-strand preparations. Error
bars indicate the upper and lower ranges of the
measured ratios.
42
Beta globin replicator
FIG. 2. Identification of two nonoverlapping,
independent replicators within the human -globin
IR. (A) A schematic representation of the IR from
the human globin locus. Replication initiates
from the region between the two adult -globin
genes (top line). The IR (IR) encompasses the
promoter and the majority of the -like-globin
gene (second line) preliminary analysis
identified a core central region within the IR,
which was essential but not sufficient for
initiation (third line) (1). The present analysis
divided the IR into two fragments designated
bGRep-P and bGRep-I (bottom line). The gray boxes
on the top line represent the globin genes white
boxes in the second line represent exons, while
the dashed line represents the direction of
transcription. (B to D) DNA fragments originating
from the human -globin IR were inserted into the
FRT site in CV-1 E25B4 cells as described. The
abundance of IR-derived DNA sequences in short,
exonuclease-resistant, newly replicated DNA was
determined by real-time quantitative PCR and
quantified as shown in panel F. A dissection of
the locus at the NcoI site (coordinate 62187)
preserved the ability to initiate DNA replication
in both fragments when each fragment was inserted
at the ectopic site, suggesting that both
fragments can function as independent
nonoverlapping replicators. Real-time PCR
analysis of the entire IR (B).
43
Beta globin replicator
Fig 2. Analysis of bGRep-P, (C), and analysis of
bGRep-I (D) are shown. Data are represented as
the number of molecules amplified from RNA-primed
nascent strands divided by the number of
molecules amplified from the same preparation by
the lacZ primers. Each histogram bar depicts the
average of three independent measurements. Error
bars represent the range of measured ratios.
44
Beta globin replicator
FIG. 3. An evolutionarily conserved alternate
AT-rich stretch is not essential for replicator
activity within the -globin Rep-P replicator. (A)
Insertion of the IR Rep-P fragment into an
FRT-containing acceptor site in the simian genome
using site-specific recombination. The insertion
vector, used to clone putative replicator
candidates, contains an FRT and a hygromycin
resistance marker (hyg). The acceptor site has an
identical FRT sequence inserted into the simian
genome. Transfection of the vector into cells
containing the target in the presence of excess
FLP recombinase leads to frequent integrations of
the entire insertion vector into the target.
Integration disrupts the expression of the lacZ
marker. Recombinant clones are selected based on
hygromycin resistance and lack of lacZ
expression, and Southern hybridization and PCR
analyses are then used to verify that
recombinant colonies contained single copies of
the insertion vector in the acceptor sites. The
filled grey arrows represent FRT sites the
double-headed arrow represents the location of
the probe used in panel B and the arrows
designated P1 to P6 represent the locations of
the primers used in panel C.
45
Beta Globin replicator
Fig 3(D) DNA fragments containing Rep-P were
mutated in vitro as indicated, inserted into the
FRT site in CV-1 E25B4 cells, and tested for
initiation activity as described in the legends
to Fig. 1 and 2. The wild-type (WT) histogram
bars show nascent-strand abundance data from the
unaltered Rep-P. The B4 bar represents the
nascent-strand abundance of a hygromycin marker
at the FRT site in the absence of sequences from
the -globin replicator. Deletion of the AT-rich
region, or replacement of this region with a
non-AT-rich linker, did not affect initiation
capacity.
46
The ? globin Replicator
Wang, L. et al. Mol. Cell. Biol. 24, 33733386
(2004).
47
Analysis of mammalian replicator

• This analysis of the beta globin IR demonstrates
  that there are no consensus sequences that can be
  found between it and the DHFR ori.
• In addition Human ORC proteins do not bind to
  specific sequences. Vashee, S. et al.
  Sequence-independent DNA binding and replication
  initiation by the human origin recognition
  complex. Genes Dev. 17, 18941908 (2003).

48
What drives ORC binding to origins

• If there are not consensus sequences that human
  ORCs bind to how do they stimulate replication?

49
What drives ORC binding to origins
Gilbert, D. M. 2004. In search of the holy
replicator. Nat. Rev. Mol. Cell. Biol. 5848-855.
Relaxed replicon model. The heterohexameric
origin-recognition complex (ORC) binds to naked
DNA indiscriminately on its own, but the
specificity of its binding to cellular chromatin
in vivo is influenced by many factors. When
presented with functionally inert (open or
indiscriminate) chromatin (a), ORC binds
nonspecifically38. However, ORC might be directed
to specific sites by (b) interacting proteins
that chaperone ORC to specific sites63 or repress
its binding to others6467. Or (c), by
superhelical tension54, which can be created by
nearby binding proteins (X) that might also
position or remove nucleosomes, or otherwise
create favourable ORC-binding sites. Superhelical
tension can also be created by transcriptional
activity, either upstream or downstream of gene
promoters69,70. ORC can also be excluded from
specific sites by (d) transcription68,71 or by
(e) general CpG DNA methylation72, which can be
quite prevalent73. Additional features of
chromatin that lie downstream of the
initiatorreplicator interaction also influence
origin specification.
50
Mammalian Replicators
Gilbert, D. M. 2004. In search of the holy
replicator. Nat. Rev. Mol. Cell. Biol. 5848-855
51
The DNA Replication fork
Garg P, Burgers PM. Crit Rev Biochem Mol Biol.
2005 40(2)115-28.

• The hierarchical scheme resulting from many
  studies is indicated by the following S.
  cerevisiae proteins and complexes
• ORC -gt Cdc6, Cdt1 -gt Mcm2-7-gt Cdc7/Dbf4 -gtMcm10,
  Dpb11/Sld2, Cdc45/Sld3, GINS, Pol e -gtRPA, Pol
  ?-primase -gt PCNA, RFC -gt Pol d

52
DNA Polymerases of Eukaryotic replication fork

• The 3 Polymerases at the Eukaryotic replication
  for are B class polymerases.
• Most B class polymerases function with circular
  clamps as a prosessivity factor

53
(No Transcript)
54
DNA Polymerase subunits
FIGURE 3 Subunit interactions in DNA polymerases.
Subunit interactions are summarized as reviewed
in detail in (MacNeill et al., 2001 Muzi-Falconi
et al., 2003 Pospiech and Syvaoja, 2003). Sizes
and names of the subunits are from S. cerevisiae,
except for Cdm1 (S. pombe), which subunit is not
found in S. cerevisiae. The polymerase subunits
are shaded in dark and the primase subunit Pri1)
of Pola in black. The third subunit Pol32) of Pol
d is extremely elongated in shape, and the
catalytic subunit of Pol e Pol 2) is a two-domain
polypeptide, interactions with the other subunits
being localized to the C-terminal domain.
55
DNA Pol ? Primase

• Hetero tetrameric enzyme.
• Structure is conserved in all eukaryotes.
• The largest subunit (Pol1) contains the DNA
  polymerase activity, but lacks exonuclease
  activity, despite the presence of an exonuclease
  domain,
• The Pri1 subunit (p48) catalyzes formation of the
  short RNA primers utilized for elongation by Pol
  ?. The remaining two subunits, the B subunit
  (Pol12, p79) and Pri2 (p58) play a role in
  stabilizing and regulating the catalytic
  subunits, and are found tightly associated with
  the polymerase and primase subunit.

56
DNA Pol ? Primase

• The primase binds the single stranded DNA
  template and catalyzes primer formation.
• eukaryotic primases, synthesis from 8 to 12
  nucleotides.
• Following the synthesis of the RNA primer, the
  Pol1 subunit of Pol ? extends the primer by
  approximately 20 nucleotides, from which lagging
  strand DNA replication continues.

57
DNA Pol ? Primase

• its activity is tightly regulated by
  post-translational modification and by
  interactions with many other proteins, from
  proteins involved in chromatin remodeling to
  replication initiation and elongation.
• Pol ? interacts with Mcm10 and Cdc45, both
  involved in initation of DNA replication.

58
DNA Polymerase ? The lagging strand

• Pol? deals efficiently with the recurring problem
  of Okazaki fragment maturation.
• Pol ? from S. cerevisiae has three subunits of
  125 (Pol3), 55 (Pol31/Hys2), and 40 kDa (Pol32)
  The enzymes from S. pombe and humans have an
  additional small fourth subunit that functions to
  stabilize the complex (see above).
• The catalytic and the second subunit form a
  stable complex, to which the third subunit is
  tethered solely via interactions with the second
  subunit.
• The third subunit of Pol ? is extremely elongated
  in shape and may be involved in higher order
  structure.
• The S. cerevisiae POL32 gene is dispensible for
  growth, although deletion mutants show poor
  growth, are sensitive to replication inhibitors
  and DNA damage, are defective for mutagenesis,
  and show synthetic lethality with a host of other
  genes that function in DNA metabolism.
• The orthologous S. pombe Cdc27 gene is essential
  for growth

59
DNA Polymerase ? The lagging strand

• The processivity clamp PCNA was first discovered
  as an auxiliary factor for the two-subunit Pol ?.
• In the absence of DNA, direct interactions
  between PCNA and Pol3/Pol31 are negligible,
• However are very strong when PCNA encircles the
  DNA.

60
DNA Polymerase ?

• it was first isolated as a multipolypeptide
  complex by Sugino and coworkers in 1990. Most
  progress has been made with the enzyme
• It is a heterotetramer of the Pol2 (256 kDa),
• Dpb2 (78 kDa), Dpb3 (23 kDa), and Dpb4 (22 kDa)
• it is likely that Pol ? is also at least a
  four-subunit enzyme in other organisms due to
  conservation of smaller subunits

61
DNA Polymerase ?

• It is thought that the C-terminus of Pol ?
  participates as an essential component in the
  assembly of the replication complex at origins.
• Pol ? loads onto origin complexes prior to primer
  synthesis, i.e., that a non-polymerase function
  of Pol ? is involved in assembly.
• Also has a double-stranded DNA binding domain in
  Pol ?.

62
DNA Polymerase ?

• PCNA stimulates DNA synthesis by Pol ?.
• A putative PCNA-binding site localizes to aa 1193
  to 1200 of Pol2.
• Deletion conferred essentially no growth defects,
  but strong damage sensitivity.

63
Strand synthesis

• Initiation of DNA replication is begun by Pol?-
  primase recruited onto RPA-coated single-stranded
  DNA by the MCM complex, and Cdc45, and Mcm10
  facilitates loading and the initiation of primer
  synthesis.
• Mcm10 may stimulate the switch from primase to
  DNA synthesis by Pol?.
• The switch from Pol? to Pol ? or ? is mediated by
  binding of a PCNA replication factor C (RFC)
  complex.
• primer synthesis is 30 nt (10 nt of RNA and 20
  nt of DNA)
• Pol?-primase is dissociated from the DNA.

64
Strand synthesis
FIGURE 5 Replication stages of the lagging
strand. The Pol ? PCNA switch promotes loading
of Pol ? on the leading strand not shown), and
Pol ? on the lagging strand.
65
Lagging strand synthesis

• PCNA-Pol ? rapidly elongates the Okazaki
  fragment.
• PCNA-stabilized elongation complex also contains
  FEN.
• Okazaki fragment maturation requires coordination
  between Pol ? and FEN1 action in order to produce
  and maintain nicks that are ligated by DNA ligase
  I.

66
Lagging strand synthesis
FIGURE 5 B) During elongation, FEN1 is proposed
to be loaded together with Pol ?, but it is only
activated upon encountering downstream DNA or
RNA.
67
Maturation of Okazaki Fragment

• When a replicating Pol ? complex runs into a
  doublestranded region, it displaces 2 to 3 nt of
  the downstream RNA or DNA (Figure 5C).
• Limited displacement by Pol ? is a reversible
  process. In the absence of FEN1, Pol ? degrades
  the newly replicated DNA using its 3 to 5
  exonuclease activity, in a process referred to as
  idling. This reiterative process of extension,
  followed by degradation, limits strand
  displacement to only a few nucleotides and allows
  the polymerase to effectively maintain a
  ligatable nick (Figure 6) (Garg et al., 2004).
  The reversible form of limited strand opening by
  Pol

68
Maturation of Okazaki Fragment
FIGURE 5 C In the model shown in Figure C, RPA
binds to long flaps only, thus preventing
cleavage by FEN1 and stimulating cleavage by
Dna2. The trimmed flap then becomes a substrate
for FEN1.
69
Completing

• When FEN1 is present replicating complex that
  runs into the double-stranded region, efficient
  nick translation ensues, and idling is inhibited
  (Figure 6). Indicative of the extremely tight
  coupling between Pol ? and FEN1, mostly
  mononucleotides are released during nick
  translation
• Finally, with DNA ligase I also present, the nick
  translation process can be terminated by ligase
  action, as rapidly as a few nucleotides past the
  RNA-DNA junction of an Okazaki fragment.

70
Completing
FIGURE 6 Nick maintenance by polymerase idling or
by nick translation. During Okazaki fragment
maturation, Pol ? and FEN1 go through multiple
cycles of displacement synthesis and FLAP cutting
(nick translation) until all RNA has been
degraded. In the absence of FEN1, idling
predominates.
71
Strand synthesis
FIGURE 5 Replication stages of the lagging
strand. The Pol ? PCNA switch promotes loading of
Pol ? on the DNA (leading strand not shown), and
Pol ? on the lagging strand. During elongation,
FEN1 is proposed to be loaded together with Pol
?, but it is only activated upon encountering
downstream DNA or RNA. In the model shown in
Figure C, RPA binds to long flaps only, thus
preventing cleavage by FEN1 and stimulating
cleavage by Dna2. The trimmed flap then becomes a
substrate for FEN1.
72
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