Roger Woodgate

The Y-Family of DNA Polymerases

We would like to thank Tomoo Ogi for generating the unrooted phylogenetic tree shown in Figure 1Figure 1 and Junetsu Ito for his comments on our proposal.

Crystal Structure of a Y-Family DNA Polymerase in Action: A Mechanism for Error-Prone and Lesion-Bypass Replication

Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) is a DinB homolog that belongs to the recently described Y-family of DNA polymerases, which are best characterized by their low-fidelity synthesis on undamaged DNA templates and propensity to traverse normally replication-blocking lesions. Crystal structures of Dpo4 in ternary complexes with DNA and an incoming nucleotide, either correct or incorrect, have been solved at 1.7 A and 2.1 A resolution, respectively. Despite a conserved active site and a hand-like configuration similar to all known polymerases, Dpo4 makes limited and nonspecific contacts with the replicating base pair, thus relaxing base selection. Dpo4 is also captured in the crystal translocating two template bases to the active site at once, suggesting a possible mechanism for bypassing thymine dimers.

Identification of additional genes belonging to the LexA regulon in Escherichia coli

Exposure of Escherichia coli to a variety of DNA‐damaging agents results in the induction of the global ‘SOS response’. Expression of many of the genes in the SOS regulon are controlled by the LexA protein. LexA acts as a transcriptional repressor of these unlinked genes by binding to specific sequences (LexA boxes) located within the promoter region of each LexA‐regulated gene. Alignment of 20 LexA binding sites found in the E. coli chromosome reveals a consensus of 5′‐TACTG(TA)5CAGTA‐3′. DNA sequences that exhibit a close match to the consensus are said to have a low heterology index and bind LexA tightly, whereas those that are more diverged have a high heterology index and are not expected to bind LexA. By using this heterology index, together with other search criteria, such as the location of the putative LexA box relative to a gene or to promoter elements, we have performed computational searches of the entire E. coli genome to identify novel LexA‐regulated genes. These searches identified a total of 69 potential LexA‐regulated genes/operons with a heterology index of < 15 and included all previously characterized LexA‐regulated genes. Probes were made to the remaining genes, and these were screened by Northern analysis for damage‐inducible gene expression in a wild‐type lexA+ cell, constitutive expression in a lexA(Def) cell and basal expression in a non‐inducible lexA(Ind−) cell. These experiments have allowed us to identify seven new LexA‐regulated genes, thus bringing the present number of genes in the E. coli LexA regulon to 31. The potential function of each newly identified LexA‐regulated gene is discussed.

Roles of E. coli DNA polymerases IV and V in lesion-targeted and untargeted SOS mutagenesis.

The expression of the Escherichia coli DNA polymerases pol V (UmuD′ 2C complex) and pol IV (DinB) increases in response to DNA damage. The induction of pol V is accompanied by a substantial increase in mutations targeted at DNA template lesions in a process called SOS-induced error-prone repair. Here we show that the common DNA template lesions, TT (6–4) photoproducts, TT cis–syn photodimers and abasic sites, are efficiently bypassed within 30 seconds by pol V in the presence of activated RecA protein (RecA*), single-stranded binding protein (SSB) and pol IIIs processivity β,γ-complex. There is no detectable bypass by either pol IV or pol III on this time scale. A mutagenic ‘signature’ for pol V is its incorporation of guanine opposite the 3′-thymine of a TT (6–4) photoproduct, in agreement with mutational spectra. In contrast, pol III and pol IV incorporate adenine almost exclusively. When copying undamaged DNA, pol V exhibits low fidelity with error rates of around 10-3 to 10-4, with pol IV being 5- to 10-fold more accurate. The effects of RecA protein on pol V, and β,γ-complex on pol IV, cause a 15,000- and 3,000-fold increase in DNA synthesis efficiency, respectively. However, both polymerases exhibit low processivity, adding 6 to 8 nucleotides before dissociating. Lesion bypass by pol V does not require β,γ-complex in the presence of non-hydrolysable ATPγS, indicating that an intact RecA filament may be required for translesion synthesis.

Y-family DNA polymerases and their role in tolerance of cellular DNA damage

The past 15 years have seen an explosion in our understanding of how cells replicate damaged DNA and how this can lead to mutagenesis. The Y-family DNA polymerases lie at the heart of this process, which is commonly known as translesion synthesis. This family of polymerases has unique features that enable them to synthesize DNA past damaged bases. However, as they exhibit low fidelity when copying undamaged DNA, it is essential that they are only called into play when they are absolutely required. Several layers of regulation ensure that this is achieved.

What a difference a decade makes: Insights into translesion DNA synthesis

Living organisms are continually under attack from a vast array of DNA-damaging agents that imperils their genomic integrity. As a consequence, cells posses an army of enzymes to repair their damaged chromosomes. However, DNA lesions often persist and pose a considerable threat to survival, because they can block the cells replicase and its ability to complete genome duplication. It has been clear for many years that cells must possess a mechanism whereby the DNA lesion could be tolerated and physically bypassed. Yet it was only within the past decade that specialized DNA polymerases for “translesion DNA synthesis” or “TLS” were identified and characterized. Many of the TLS enzymes belong to the recently described “Y-family” of DNA polymerases. By possessing a spacious preformed active site, these enzymes can physically accommodate a variety of DNA lesions and facilitate their bypass. Flexible DNA-binding domains and a variable binding pocket for the replicating base pair further allow these TLS polymerases to select specific lesions to bypass and favor distinct non-Watson–Crick base pairs. Consequently, TLS polymerases tend to exhibit much lower fidelity than the cells replicase when copying normal DNA, which results in a dramatic increase in mutagenesis. Occasionally this can be beneficial, but it often speeds the onset of cancer in humans. Cells use both transcriptional and posttranslational regulation to keep these low-fidelity polymerases under strict control and limit their access to a replication fork. Our perspective focuses on the mechanistic insights into TLS by the Y-family polymerases, how they are regulated, and their effects on genomic (in)stability that have been described in the past decade.

Molecular Analysis of Antibiotic Resistance Gene Clusters in Vibrio cholerae O139 and O1 SXT Constins

ABSTRACT Many recent Asian clinical Vibrio cholerae E1 Tor O1 and O139 isolates are resistant to the antibiotics sulfamethoxazole (Su), trimethoprim (Tm), chloramphenicol (Cm), and streptomycin (Sm). The corresponding resistance genes are located on large conjugative elements (SXT constins) that are integrated into prfC on the V. cholerae chromosome. We determined the DNA sequences of the antibiotic resistance genes in the SXT constin in MO10, an O139 isolate. In SXTMO10, these genes are clustered within a composite transposon-like structure found near the elements 5′ end. The genes conferring resistance to Cm (floR), Su (sulII), and Sm (strA and strB) correspond to previously described genes, whereas the gene conferring resistance to Tm, designated dfr18, is novel. In some other O139 isolates the antibiotic resistance gene cluster was found to be deleted from the SXT-related constin. The El Tor O1 SXT constin, SXTET, does not contain the same resistance genes as SXTMO10. In this constin, the Tm resistance determinant was located nearly 70 kbp away from the other resistance genes and found in a novel type of integron that constitutes a fourth class of resistance integrons. These studies indicate that there is considerable flux in the antibiotic resistance genes found in the SXT family of constins and point to a model for the evolution of these related mobile elements.

Misinsertion and bypass of thymine–thymine dimers by human DNA polymerase ι

Human DNA polymerase ι (polι) is a recently discovered enzyme that exhibits extremely low fidelity on undamaged DNA templates. Here, we show that polι is able to facilitate limited translesion replication of a thymine–thymine cyclobutane pyrimidine dimer (CPD). More importantly, however, the bypass event is highly erroneous. Gel kinetic assays reveal that polι misinserts T or G opposite the 3′ T of the CPD ∼1.5 times more frequently than the correct base, A. While polι is unable to extend the T·T mispair significantly, the G·T mispair is extended and the lesion completely bypassed, with the same efficiency as that of the correctly paired A·T base pair. By comparison, polι readily misinserts two bases opposite a 6‐4 thymine–thymine pyrimidine–pyrimidone photoproduct (6‐4PP), but complete lesion bypass is only a fraction of that observed with the CPD. Our data indicate, therefore, that polι possesses the ability to insert nucleotides opposite UV photoproducts as well as to perform unassisted translesion replication that is likely to be highly mutagenic.

MSH2–MSH6 stimulates DNA polymerase η, suggesting a role for A:T mutations in antibody genes

Activation-induced cytidine deaminase deaminates cytosine to uracil (dU) in DNA, which leads to mutations at C:G basepairs in immunoglobulin genes during somatic hypermutation. The mechanism that generates mutations at A:T basepairs, however, remains unclear. It appears to require the MSH2–MSH6 mismatch repair heterodimer and DNA polymerase (pol) η, as mutations of A:T are decreased in mice and humans lacking these proteins. Here, we demonstrate that these proteins interact physically and functionally. First, we show that MSH2–MSH6 binds to a U:G mismatch but not to other DNA intermediates produced during base excision repair of dUs, including an abasic site and a deoxyribose phosphate group. Second, MSH2 binds to pol η in solution, and endogenous MSH2 associates with the pol in cell extracts. Third, MSH2–MSH6 stimulates the catalytic activity of pol η in vitro. These observations suggest that the interaction between MSH2–MSH6 and DNA pol η stimulates synthesis of mutations at bases located downstream of the initial dU lesion, including A:T pairs.

Localization of DNA polymerases eta and iota to the replication machinery is tightly co-ordinated in human cells.

Y‐family DNA polymerases can replicate past a variety of damaged bases in vitro but, with the exception of DNA polymerase η (polη), which is defective in xeroderma pigmentosum variants, there is little information on the functions of these polymerases in vivo. Here, we show that DNA polymerase ι (polι), like polη, associates with the replication machinery and accumulates at stalled replication forks following DNA‐damaging treatment. We show that polη and polι foci form with identical kinetics and spatial distributions, suggesting that localization of these two polymerases is tightly co‐ordinated within the nucleus. Furthermore, localization of polι in replication foci is largely dependent on the presence of polη. Using several different approaches, we demonstrate that polη and polι interact with each other physically and that the C‐terminal 224 amino acids of polι are sufficient for both the interaction with polη and accumulation in replication foci. Our results provide strong evidence that polη targets polι to the replication machinery, where it may play a general role in maintaining genome integrity as well as participating in translesion DNA synthesis.