STUDIES ON DNA REPLICATION, REPAIR, AND MUTAGENESIS IN EUKARYOTIC AND PROKARYOTIC CELLS
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Roger
Woodgate, PhD, Head,
Section on DNA Replication, Repair and Mutagenesis Ekaterina
Chumakov, PhD, Staff Scientist Alexandra
Vaisman, PhD, Senior Research Fellow Mary
McLenigan, BS, Chemist John McDonald,
PhD, Biologist Samantha
Mead, PhD, Visiting Fellow Antonio
Vidal, PhD, Visiting Fellow Brian
Plosky, PhD, Postdoctoral Fellow Majda
Valjavec-Gratian, PhD, Postdoctoral Fellow Amie Gupta, Student |
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Under optimal conditions, the fidelity
of DNA replication is extremely high: it is estimated that, on average, only
one error occurs in every 1010 bases replicated. However, as
living organisms are continually subjected to a variety of endogenous and
exogenous DNA-damaging agents, optimal conditions rarely prevail in vivo.
Even though all organisms have evolved elaborate repair pathways to deal with
DNA damage, the repair pathways rarely operate with 100 percent efficiency;
as a consequence, the persisting DNA lesions are replicated, but with much
lower fidelity than undamaged DNA. Our general aim therefore is to understand
the molecular mechanisms by which mutations are introduced into damaged DNA.
The process is commonly referred to as translesion DNA synthesis (TLS) or
translesion replication (TR) and is thought to be facilitated by one or more
of the Y-family of DNA polymerases that are phylogenetically conserved from
bacteria to humans. As in past years, we continue to investigate the TLS
process in all three kingdoms of life: bacteria, archaea, and eukaryotes. Translesion
replication in prokaryotes Gupta, Mead,
Valjavec-Gratian, Woodgate; in collaboration with Goodman, Paetzel In E.
coli, efficient translesion replication of many DNA lesions occurs only
when the UmuC protein physically interacts with a dimer of UmuDī to form a
heterotrimeric complex of UmuDī2C, (known as E. coli pol
V). Because pol V is a low-fidelity enzyme, its activities within the cell
are strictly controlled at several levels. For example, the umu genes
are arranged in an operon and are negatively regulated at the transcriptional
level by the SOS repressor LexA. Transcriptional regulation is, however,
insufficient, and the cellular levels of UmuD and UmuC are kept to a minimum
through their rapid Lon-mediated proteolytic degradation. After cellular DNA
damage, RecA protein nucleates on regions of single-stranded DNA and mediates
the post-translational self-cleavage of LexA, leading to LexA’s
inactivation and to the derepression of genes in the LexA-regulon, including umuD
and umuC. Interestingly, UmuD undergoes a mechanistically similar
self-cleavage reaction in which cleavage of its N-terminal 24 amino acids
converts it to UmuDī and activates it for its role in SOS mutagenesis. UmuD
and UmuDī both form homodimers but, when mixed, preferentially associate as a
UmuD/Dī heterodimer, with UmuDī becoming susceptible to proteolysis by the
ClpXP serine protease. Degradation of the mutagenically active UmuDī subunit
therefore helps return cells to a resting state once cellular DNA damage has
been repaired and the need for pol V has abated. In
vitro replication assays reveal that regulation of the
catalytic activity of pol V is also modulated through several protein-protein
interactions. For example, pol V does not catalyze TLS alone but is instead
an essential component of a multiprotein “mutasome” complex
composed of RecA protein, beta sliding-clamp, and single-strand DNA binding
protein (SSB). In collaboration with Myron Goodman, we have investigated in
detail the nature of the interactions between RecA and pol V. We found that
pol V and RecA physically interact through two distinguishable mechanisms.
The first occurs when pol V binds to RecA through its UmuC subunit in the
absence of DNA and ATP while the second occurs through the enzyme’s
UmuDī subunit in the presence of DNA and ATP. Pol V–catalyzed synthesis
with mutant RecAs with an increased affinity for ssDNA revealed that any
additional RecA bound to DNA inhibits normal DNA synthesis and TLS, thereby
suggesting that a RecA nucleoprotein filament is unlikely to be a
prerequisite for SOS mutagenesis. Pol V failed to synthesize DNA with a RecA
mutant (RecA1730) that is defective in promoting SOS mutagenesis in vivo,
suggesting that RecA may serve as an obligate accessory factor necessary to
stimulate pol V activity both in vitro and in vivo. Paetzel M, Woodgate R. UmuD and UmuDī proteins. In: Barrett AJ,
Rawlings ND, Woessner JF, eds. Handbook of Proteolytic Enzymes, 2d ed.
London: Academic Press, Elsevier Science, 2004;1976-1981. Shen X, Woodgate R, Goodman MF. Escherichia coli DNA
polymerase V subunit exchange: a post-SOS mechanism to curtail error-prone
DNA synthesis. J Biol Chem 2003;278:52546-52550. Translesion
replication in Saccharomyces cerevisiae McDonald, Woodgate; in
collaboration with Lawrence In
addition to a variety of mechanisms that repair damage to its genome, the
yeast Saccharomyces cerevisiae possesses mechanisms that lead to the
tolerance of DNA damage by promoting translesion replication. Genetic
experiments previously implicated DNA polymerases Eta, encoded by RAD30,
and Zeta, encoded by REV3 and REV7, as requirements for the
bypass of lesions that would otherwise cause the replicase to stall. In addition
to these enzymes, translesion replication often requires the Rev1 protein,
which appears to be essential for pol zeta activity, and Pol32p, a subunit of
DNA polymerase delta required for induced mutagenesis. The various functions
of these proteins in translesion replication remain unclear. We thus
investigated their roles in the in vivo bypass of an abasic site, 6-4
photoadduct (6-4PP),and cis-syn cyclobutane dimer (CPD) by
transforming isogenic yeast strains deleted for RAD30, REV3, REV1,
or POL32 with duplex plasmids carrying one of the DNA lesions within a
28-nucleotide single-stranded region. Bypass frequencies and nucleotide
insertion spectra revealed by the experiments showed that pol eta is only
rarely involved in the bypass of the abasic sites or 6-4PP but was, as
expected, solely responsible for the bypass of the CPD. The insertion of dG
opposite the 3īT of the 6-4PP, characteristic of pol eta, was significantly
lower (4 percent) in the rad30 deletion strain than all insertions in
the wild type (10 percent), showing some involvement of pol eta in such
events. However, the results also suggest that another enzyme can generate
the mutations; thus, we hypothesize that pol zeta is responsible for
insertion in all other bypass events. Pol delta is also clearly involved in
mutagenesis, given that strains lacking Pol32 are known to be deficient in
mutagenesis; in keeping with this observation, a pol32 deletion strain
shows as little bypass of the abasic sites or 6-4PP photoadduct as those
deficient in pol zeta or Rev1. Thus, TLS in Saccharomyces cerevisiae
is complex and appears often to require a combination of one or more
polymerases to facilitate the bypass of many DNA adducts. Gibbs PEM, McDonald JP, Woodgate R, Structural
analysis of lesion bypass Plosky, Vaisman,
Woodgate; in collaboration with Holliger, Kunkel, Yang We
previously identified and cloned a Y-family DNA polymerase from the
thermostable archaeon Sulfolobus solfataricus P2, which we called DNA
polymerase IV (Dpo4). In collaboration with Wei Yang, we solved, by X-ray
diffraction, the crystal structure of Dpo4 in a ternary complex with an
undamaged DNA template and an incoming dexoyribose nucleoside triphosphate,
revealing that, like all DNA polymerases, Dpo4 possesses a topology similar
to a right hand with protein domains that resemble “fingers,” a
“palm,” and a “thumb.” However, these domains are all
stubby, and the active site of the enzyme is large and exposed to solvent.
Dpo4 also possesses a unique domain called the “little finger,”
which helps the enzyme bind to DNA. We previously hypothesized that the
“cavernous” active site of Dpo4 is large enough to accept bulky
DNA adducts that normally represent a block to replication by high-fidelity
polymerases with a more restrictive active site. Indeed, we recently
crystallized benzo[a]pyrene diol epoxide (BPDE), a large polycyclic
aromatic hydrocarbon, in the active site of Dpo4. We solved two conformations
of the BPDE lesion, one in which the lesion was intercalated between base
pairs and another in which it was solvent-exposed in the major groove.
Organic solvents that reduce the dielectric constant and appear more
favorable for DNA replication by Dpo4 can stabilize the latter conformation.
Our structures therefore provide mechanistic insights into how cells generate
mutations during replication of DNA containing carcinogen-induced BPDE
adducts. Another
lesion crystallized in the active site of Dpo4 was an abasic site. Even
though the lesion is small, it nevertheless blocks replication by cellular
replicases, as the base is physically absent and there is no genetic
information to instruct continued DNA synthesis. We determined the crystal
structures of Dpo4 complexed with five different abasic site–containing
DNA substrates and discovered that translesion synthesis is
template-directed, with the abasic site looping out, and that the incoming
nucleotide is opposite the base 5ī to the lesion. The ensuing DNA synthesis
generates a –1 frameshift when the abasic site remains extra-helical.
We also observed that template realignment during primer extension resulted
in base substitutions or even +1 frameshifts. In the case of a +1 frameshift,
the extra nucleotide was accommodated in the solvent-exposed minor groove. In
addition, we crystallized an unproductive Dpo4 ternary complex, which
suggested that the flexible little finger (LF) domain facilitates DNA
orientation and translocation during translesion synthesis. We
further investigated the possibility of orientation/translocation by making
chimeras of Dpo4 and Dbh in which their LF domains had been interchanged.
Dpo4 and Dbh originate from two closely related strains of Sulfolobaceae,
yet the two polymerases exhibit different enzymatic properties in vitro.
For example, Dpo4 can replicate past a variety of DNA lesions, but Dbh does
so with a much lower efficiency. When replicating undamaged DNA, Dpo4 is
prone to make base-pair substitutions while Dbh predominantly makes
single-base deletions. Interestingly, by replacing the LF domain of Dbh with
that of Dpo4, the enzymatic properties of the chimeric enzyme are more
Dpo4-like in that the enzyme is more processive, is able to bypass an abasic
site and a CPD, and predominantly makes base-pair substitutions when
replicating undamaged DNA. The converse holds for the Dpo4LFDbh chimera,
which is more Dbh-like in its processivity and ability to bypass DNA adducts
and generate single-base deletion errors. Our studies therefore indicate that
the unique but variable LF domain of Y-family polymerases plays a major role
in determining the enzymatic and biological properties of each Y-family
member. We
have also investigated how replicative DNA polymerases recognize their
substrates with such high specificity. In a collaborative study with Philipp
Holliger, we developed a strategy to expand the substrate range of
polymerases by simply selecting for polymerases that were able to extend a
distorting 3ī mismatch. We obtained several mutants of Taq DNA polymerase
that not only extended mismatches promiscuously but that had also acquired a
generic ability to process a diverse range of noncanonical substrates while
maintaining high catalytic turnover, processivity, and fidelity.
Interestingly, unlike the wild-type Taq enzyme, the mutant enzymes also
bypassed blocking lesions such as an abasic site, a CPD, or the base analog
5-nitroindol. We also performed PCR amplification with complete substitution
of all four nucleotide triphosphates with phosphorothioates or the
substitution of one with the equivalent fluorescent dye–labeled
nucleotide triphosphate. In the latter case, we obtained PCR products that
had a 20-fold greater fluorescence intensity than those generated with
wild-type Taq polymerase. Boudsocq F, Kokoska RJ, Plosky BS, Vaisman A, Hong Ling H,
Kunkel TA, Yang W, Woodgate R. Investigating the role of the little finger
domain of Y-family DNA polymerases in low-fidelity synthesis and translesion
replication. J Biol Chem 2004;279:32932-32940. Ghadessy FJ, Boudsocq F, Loakes D, Brown A, Iwai S, Vaisman A,
Woodgate R, Holliger P. Expansion of substrate tolerance in an A-family DNA
polymerase by directed evolution. Nat Biotech 2004;22:755-759. Ling H, Boudsocq F, Plosky BS, Woodgate R, Yang W. Replication of
a cis-syn thymine dimer at atomic resolution. Nature 2003;424:1083-1087. Ling H, Boudsocq F, Woodgate R, Yang W. Snapshots of replication
through an abasic lesion; structural basis for base substitutions and
frameshifts. Mol Cell 2004;13:751-762. Ling H, Sayer JM, Plosky BS, Yagi H, Boudsocq F, Woodgate R,
Jerina DM, Yang W. Crystal structure of a benzo[alpha]pyrene diol
epoxide adduct in a ternary complex with a DNA polymerase. Proc Natl Acad
Sci USA 2004;101:2265-2269. Characterization
of human DNA polymerase iota Chumakov, McDonald,
McLenigan, Mead, Vidal, Vaisman, Woodgate; in collaboration with Hanaoka,
Kunkel, Lehmann Humans
possess four Y-family polymerases: pol eta, pol iota, pol kappa, and Rev1.
Pol iota is of particular interest, as it was originally identified by
scientists within our laboratory. Pol iota is a paralog of pol eta but,
unlike pol eta, which efficiently and accurately bypasses UV-induced CPDs,
the ability of pol iota to bypass CPDs is somewhat varied, with results ranging
from limited misinsertion opposite CPDs to complete lesion bypass. While
defects in pol eta lead to the sunlight-sensitive and cancer-prone Xeroderma
pigmentosum Variant (XP-V) phenotype, the biological function of pol iota
remains to be determined. Our recent studies reveal that human pol iota and
the proliferating cell nuclear antigen (PCNA) physically interact and that
the interaction stimulates the processivity of pol iota in a
template-dependent manner in vitro. Mutations in one of the putative
PCNA-binding motifs (PIP-box) of pol iota or the interdomain connector loop
of PCNA diminished the binding between pol iota and PCNA and concomitantly
reduced PCNA-dependent stimulation of pol iota activity. Furthermore, the
mutant pol iota-PIP box mutant failed to accumulate into replication foci
after cellular DNA damage, indicating that an interaction between pol iota
and PCNA is essential for foci formation. Based on our observations, we
hypothesize that PCNA, acting as both a scaffold and a modulator of the
different activities involved in replication, recruits and coordinates
replicative and TLS-polymerases so as to ensure genome integrity. Plosky BS, Woodgate R. Switching from high-fidelity replicases
to low-fidelity lesion-bypass polymerases. Curr Opin Genet Dev
2004;14:113-119. Vaisman A, Frank EG, Iwai S, Ohashi E, Ohmori H, Hanaoka F,
Woodgate R. Sequence context-dependent replication of DNA templates
containing UV-induced lesions by human DNA polymerase iota. DNA Repair
2003;2:991-1006. Vaisman A, Vaisman A, Woodgate R. Translesion DNA polymerases, eukaryotic.
In: Lennarz WJ, Lane MD, eds. Encyclopedia of Biological Chemistry, vol 4.
Vidal AE, Kannouche P, Podust VN, Yang W, Lehmann AR, Woodgate
R. PCNA-dependent coordination of the biological functions of human DNA
polymerase iota. J Biol Chem 2004;279:48360-48368. COLLABORATORS Myron F. Goodman, PhD, Fumio Hanaoka, PhD, Philipp Holliger, PhD, Medical Research
Council Laboratory for Molecular Biology, Thomas A. Kunkel, PhD, Laboratory of
Molecular Genetics, NIEHS, Christopher W. Lawrence, PhD, Alan R. Lehmann, PhD, Mark Paetzel, PhD, Wei Yang, PhD, Laboratory of Molecular
Biology, NIDDK,
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