This paper reports reconstitution of 5'-nick-directed mismatch repair using purified human proteins. The reconstituted system includes MutSalpha or MutSbeta, MutLalpha, RPA, EXO1, HMGB1, PCNA, RFC, polymerase delta, and ligase I. In this system, MutSbeta plays a limited role in repair of base-base mismatches, but it processes insertion/deletion mispairs much more efficiently than MutSalpha, which efficiently corrects both types of heteroduplexes. MutLalpha reduces the processivity of EXO1 and terminates EXO1-catalyzed excision upon mismatch removal. In the absence of MutLalpha, mismatch-provoked excision by EXO1 occurs extensively. RPA and HMGB1 play similar but complementary roles in stimulating MutSalpha-activated, EXO1-catalyzed excision in the presence of a mismatch, but RPA has a distinct role in facilitating MutLalpha-mediated excision termination past mismatch. Evidence is provided that efficient repair of a single mismatch requires multiple molecules of MutSalpha-MutLalpha complex. These data suggest a model for human mismatch repair involving coordinated initiation and termination of mismatch-provoked excision.

Error-free lesion bypass and error-prone lesion bypass are important cellular responses to DNA damage during replication, both of which require a DNA polymerase (Pol). To identify lesion bypass DNA polymerases, we have purified human Polkappa encoded by the DINB1 gene and examined its response to damaged DNA templates. Here, we show that human Polkappa is a novel lesion bypass polymerase in vitro. Purified human Polkappa efficiently bypassed a template 8-oxoguanine, incorporating mainly A and less frequently C opposite the lesion. Human Polkappa most frequently incorporated A opposite a template abasic site. Efficient further extension required T as the next template base, and was mediated mainly by a one-nucleotide deletion mechanism. Human Polkappa was able to bypass an acetylaminofluorene-modified G in DNA, incorporating either C or T, and less efficiently A opposite the lesion. Furthermore, human Polkappa effectively bypassed a template (-)-trans-anti-benzo[a]pyrene-N:(2)-dG lesion in an error-free manner by incorporating a C opposite the bulky adduct. In contrast, human Polkappa was unable to bypass a template TT dimer or a TT (6-4) photoproduct, two of the major UV lesions. These results suggest that Polkappa plays an important role in both error-free and error-prone lesion bypass in humans.

DNA polymerase activity is essential for replication, recombination, repair, and mutagenesis. All DNA polymerases studied so far from any biological source synthesize DNA by the Watson-Crick base-pairing rule, incorporating A, G, C, and T opposite the templates T, C, G, and A, respectively. Non-Watson-Crick base pairs would lead to mutations. In this report, we describe the ninth human DNA polymerase, Pol, encoded by the RAD30B gene. We show that human Pol violates the Watson-Crick base-pairing rule opposite template T. During base selection, human Pol preferred T-G base pairing, leading to G incorporation opposite template T. The resulting T-G base pair was less efficiently extended by human Pol compared to the Watson-Crick base pairs. Consequently, DNA synthesis frequently aborted opposite template T, a property we designated the T stop. This T stop restricted human Pol to a very short stretch of DNA synthesis. Furthermore, kinetic analyses show that human Pol copies template C with extraordinarily low fidelity, misincorporating T, A, and C with unprecedented frequencies of 1/9, 1/10, and 1/11, respectively. Human Pol incorporated one nucleotide opposite a template abasic site more efficiently than opposite a template T, suggesting a role for human Pol in DNA lesion bypass. The unique features of preferential G incorporation opposite template T and T stop suggest that DNA Pol may additionally play a specialized function in human biology.DNA synthesis is catalyzed by a DNA polymerase (Pol). In humans, eight DNA polymerases have been identified thus far: -, -, and -(6, 10, 15, 17, 19, 32). All DNA polymerases studied so far from any biological source synthesize DNA by the Watson-Crick base-pairing rule, incorporating A, G, C, and T opposite the templates T, C, G, and A, respectively (37). Nuclear DNA replication involves Pol␣, -␦, and -ε, while mitochondrial DNA replication requires Pol␥ (15). Pol␤ is a major repair synthesis enzyme during base excision repair (16). Pol is believed to be involved in the damage-induced mutagenesis pathway for translesion DNA synthesis (6,17,27). Pol, encoded by the XPV gene, is a DNA lesion bypass polymerase capable of both error-free and errorprone translesion synthesis using several damaged DNA templates (11,19,39). Pol is predicted to be involved in repair of DNA interstrand cross-links (32).Recently, four families of proteins have been identified as forming the UmuC superfamily (4, 23). The prototypic members of this superfamily are the Escherichia coli UmuC, E. coli DinB, yeast Saccharomyces cerevisiae Rev1, and S. cerevisiae Rad30. UmuC is a subunit of the E. coli DNA polymerase V, a polymerase required in the damage-induced mutagenesis pathway (30,33). DinB is the E. coli DNA polymerase IV (34), which is involved in untargeted mutagenesis (1, 13, 34). Rev1 is a dCMP transferase, that is required in the damage-induced mutagenesis (26). Rad30 is the yeast DNA Pol (11). Human homologues of DinB (5, 28), Rev1 (7, 18), and Rad30 (10, 20, 23) have been isolated. In humans, two R...

The effectiveness of in vitro primer elongation reactions catalyzed by human bypass DNA polymerases (hDinB1), pol (hRad30A), pol (hRad30B), and yeast pol (Rev3 and Rev7) in site-specifically modified template oligonucleotide strands were studied in vitro. The templates contained single bulky lesions derived from the trans-addition of the mutagenic (؉)-or (؊)-enantiomers of r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (a metabolite of the environmental carcinogen benzo[a]pyrene), to the exocyclic amino groups of guanine or adenine in oligonucleotide templates 33, or more, bases long. In "running start" primer extension reactions, pol effectively bypassed both the stereoisomeric (؉)-and (؊)-trans-guanine adducts but not the analogous adenine adducts. In sharp contrast, pol , which exhibits considerable sequence homology with pol (both belong to the group of Y family polymerases), is partially blocked by the guanine adducts and the (؊)-trans-adenine adduct, although the stereoisomeric (؉)-trans-adenine adduct is more successfully bypassed. Neither pol nor pol , either alone or in combination, were effective in trans-lesion synthesis past the same adducts. In all cases, the fidelity of insertion is dependent on adduct stereochemistry and structure. Generally, error-free nucleotide insertion opposite the lesions tends to depend more on adduct stereochemistry than error-prone insertion. None of the polymerases tested are a universal bypass polymerase for the stereoisomeric bulky polycyclic aromatic hydrocarbon-DNA adducts derived from anti-BPDE.Several new prokaryotic and eukaryotic DNA polymerases (pol) 1 involved in trans-lesional synthesis have been recently identified (reviewed in Refs. 1-5). The functional characteristics of the UmuC/DinB/Rev1p/Rad30 DNA polymerases belonging to the Y family of DNA polymerases (5) have been extensively investigated (6 -11). These polymerases are able to bypass a variety of DNA lesions in vitro (8,(12)(13)(14)(15)(16)(17)(18)(19)(20) that are known to block replication catalyzed by "classical" polymerases such as pol ␣, pol ␤, phage T7, the Klenow fragment of pol I from Escherichia coli, and human immunodeficiency virusreverse transcriptase, etc. (see Refs. 21-18 for example).The efficiencies and fidelities of trans-lesion synthesis catalyzed by Y family polymerases vary significantly among the different subfamilies and also depend remarkably on the type of DNA damage. Purified human pol (DinB1, 870 amino acids in size) efficiently bypasses (Ϫ)-trans-[BP]-N 2 -dG adducts (bulky lesions derived from the binding of the mutagen r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene ((ϩ)-or (Ϫ)-anti-BPDE) to the exocyclic amino groups of guanine and adenine residues in DNA (Scheme 1) with the insertion of a C opposite the lesion (12). A truncated form of pol , a 560-amino acid form pol (⌬C), missing motifs VIIa and VIIb and obtained from a baculovirus expression system, also bypasses the (ϩ)- trans-[BP]-N2 -dG adduct (29). Human pol also bypasses 8-oxo-dG lesio...