Haruo Ohmori

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.

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.

Interaction of hREV1 with three human Y-family DNA polymerases

Polκ is one of many DNA polymerases involved in translesion DNA synthesis (TLS). It belongs to the Y‐family of polymerases along with Polη, Polι and hREV1. Unlike Polη encoded by the xeroderma pigmentosum variant (XPV) gene, Polκ is unable to bypass UV‐induced DNA damage in vitro, but it is able to bypass benzo[a]pyrene (B[a]P)‐adducted guanines accurately and efficiently. In an attempt to identify factor(s) targeting Polκ to its cognate DNA lesion(s), we searched for Polκ‐interacting proteins by using the yeast two‐hybrid assay. We found that Polκ interacts with a C‐terminal region of hREV1. Polη and Polι were also found to interact with the same region of hREV1. The interaction between Polκ and hREV1 was confirmed by pull‐down and co‐immunoprecipitation assays. The C‐terminal region of hREV1 is known to interact with hREV7, a non‐catalytic subunit of Polζ that is another structurally unrelated TLS enzyme, and we show that Polκ and hREV7 bind to the same C‐terminal region of hREV1. Thus, our results suggest that hREV1 plays a pivotal role in the multi‐enzyme, multi‐step process of translesion DNA synthesis.

Fidelity and processivity of DNA synthesis by DNA polymerase κ, the product of the human DINB1 gene

Mammalian DNA polymerase κ (pol κ), a member of the UmuC/DinB nucleotidyl transferase superfamily, has been implicated in spontaneous mutagenesis. Here we show that human pol κ copies undamaged DNA with average single-base substitution and deletion error rates of 7 × 10−3 and 2 × 10−3, respectively. These error rates are high when compared to those of most other DNA polymerases. pol κ also has unusual error specificity, producing a high proportion of T·CMP mispairs and deleting and adding non-reiterated nucleotides at extraordinary rates. Unlike other members of the UmuC/DinB family, pol κ can processively synthesize chains of 25 or more nucleotides. This moderate processivity may reflect a contribution of C-terminal residues, which include two zinc clusters. The very low fidelity and moderate processivity of pol κ is novel in comparison to any previously studied DNA polymerase, and is consistent with a role in spontaneous mutagenesis.

Polκ protects mammalian cells against the lethal and mutagenic effects of benzo[a]pyrene

Several low-fidelity DNA polymerases have recently been discovered that are able to bypass DNA lesions during DNA synthesis in vitro. The efficiency and accuracy of lesion bypass is, however, both polymerase and lesion specific. For example, in vitro studies revealed that human DNA polymerase κ (Polκ) is unable to insert a base opposite a cis-syn thymine-thymine dimer or cisplatin adduct, yet can bypass some DNA lesions such as abasic site and acetylaminofluorene-adducted guanine in an error-prone manner. More importantly, Polκ is able to bypass benzo[a]pyrene (B[a]P)-adducted guanine accurately and efficiently. To investigate the biological function of Polκ, we have generated mouse embryonic stem (ES) cells deficient in the Polk gene encoding the enzyme. Polk-deficient ES cells grow normally and their sensitivities to UV and x-ray radiation are only slightly affected. In contrast, the mutant cells are highly sensitive to both killing and mutagenesis induced by B[a]P. Furthermore, the spectrum of mutations recovered in the Polk-deficient cells is different from that in the wild-type cells. Thus, our results indicate that Polκ plays an important role in suppressing mutations at DNA lesions generated by B[a]P.

Rad18 Regulates DNA Polymerase κ and Is Required for Recovery from S-Phase Checkpoint-Mediated Arrest

ABSTRACT We have investigated mechanisms that recruit the translesion synthesis (TLS) DNA polymerase Polκ to stalled replication forks. The DNA polymerase processivity factor PCNA is monoubiquitinated and interacts with Polκ in cells treated with the bulky adduct-forming genotoxin benzo[a]pyrene dihydrodiol epoxide (BPDE). A monoubiquitination-defective mutant form of PCNA fails to interact with Polκ. Small interfering RNA-mediated downregulation of the E3 ligase Rad18 inhibits BPDE-induced PCNA ubiquitination and association between PCNA and Polκ. Conversely, overexpressed Rad18 induces PCNA ubiquitination and association between PCNA and Polκ in a DNA damage-independent manner. Therefore, association of Polκ with PCNA is regulated by Rad18-mediated PCNA ubiquitination. Cells from Rad18−/− transgenic mice show defective recovery from BPDE-induced S-phase checkpoints. In Rad18−/− cells, BPDE induces elevated and persistent activation of checkpoint kinases, indicating persistently stalled forks due to defective TLS. Rad18-deficient cells show reduced viability after BPDE challenge compared with wild-type cells (but survival after hydroxyurea or ionizing radiation treatment is unaffected by Rad18 deficiency). Inhibition of RPA/ATR/Chk1-mediated S-phase checkpoint signaling partially inhibited BPDE-induced PCNA ubiquitination and prevented interactions between PCNA and Polκ. Taken together, our results indicate that ATR/Chk1 signaling is required for Rad18-mediated PCNA monoubiquitination. Recruitment of Polκ to ubiquitinated PCNA enables lesion bypass and eliminates stalled forks, thereby attenuating the S-phase checkpoint.

Mutation enhancement by DINB1, a mammalian homologue of the Escherichia coli mutagenesis protein dinB.

The Escherichia coli dinB gene is an SOS gene known to be required for λ phage untargeted mutagenesis. When over‐expressed, it exhibits a potent mutagenic activity without any exogenous treatment to damage DNA. Frameshift mutations at a run of identical bases are most enhanced. The product DinB is structurally related to the E. coli UmuC protein and the Saccharomyces cerevisiae Rev1 and Rad30 proteins, all of which are shown to be involved in bypass synthesis at a DNA lesion.

Structural analysis of the dnaA and dnaN genes of Escherichia coli

The nucleotide sequence of the entire region containing the Escherichia coli dnaA and dnaN genes was determined. Base substitutions by such mutations as dnaA46, dnaA167, dnaN59, and dnaN806 were also identified. Analyses of coding frames, the mutational base substitutions, and other data indicate that dnaN follows dnaA, both have the same orientation, and are separated by only 4 bp. The deduced amino acid sequence specifies Mrs and isoelectric points consistent with those of the previously identified gene products. The transcriptional initiation site of the dnaA gene was assigned by analysis of in vitro RNA products. Examination of the intercistronic sequence and analysis of in vitro transcription supported the notion that the dnaA and dnaN genes constitute a single operon.

Expression of human and mouse genes encoding polκ: testis-specific developmental regulation and AhR-dependent inducible transcription

Backgrounds Human polκ is a newly identified low‐fidelity DNA polymerase. While the enzyme bypasses an abasic site and acetylaminofluorene‐adduct in an error‐prone manner, it bypasses benzo[a]pyrene‐N2‐dG lesions in a mostly error‐free manner by incorporating predominantly dC opposite the bulky lesions. Benzo[a]pyrene (B[a]P) is activated through intracellular process mediated by the arylhydrocarbon receptor (AhR, also called the dioxin receptor), which is a ligand‐activated transcription factor with high affinities for aromatic compounds such as B[a]P and dioxin.

Inhibition of Escherichia coli RecA coprotease activities by DinI.

In Escherichia coli, the SOS response is induced upon DNA damage and results in the enhanced expression of a set of genes involved in DNA repair and other functions. The initial step, self‐cleavage of the LexA repressor, is promoted by the RecA protein which is activated upon binding to single‐stranded DNA. In this work, induction of the SOS response by the addition of mitomycin C was found to be prevented by overexpression of the dinI gene. dinI is an SOS gene which maps at 24.6 min of the E.coli chromosome and encodes a small protein of 81 amino acids. Immunoblotting analysis with anti‐LexA antibodies revealed that LexA did not undergo cleavage in dinI‐overexpressed cells after UV irradiation. In addition, the RecA‐dependent conversion of UmuD to UmuD′ (the active form for mutagenesis) was also inhibited in dinI‐overexpressed cells. Conversely, a dinI‐deficient mutant showed a slightly faster and more extensive processing of UmuD and hence higher mutability than the wild‐type. Finally, we demonstrated, by using an in vitro reaction with purified proteins, that DinI directly inhibits the ability of RecA to mediate self‐cleavage of UmuD.