The damage-inducible UmuD′ and UmuC proteins are required for most SOS mutagenesis in Escherichia coli . Our recent assay to reconstitute this process vitro , using a native 2 C complex, revealed that the highly purified preparation contained DNA polymerase activity. Here we eliminate possibility activity is caused by contaminating show it intrinsic C. E. dinB has recently been shown have (pol IV). We suggest C, fifth discovered be designated as pol V. In presence of RecA, β sliding clamp, γ...

The Saccharomyces cerevisiae RAD30 gene encodes DNA polymerase eta. Humans possess two Rad30 homologs. One (RAD30A/POLH) has previously been characterized and shown to be defective in humans with the Xeroderma pigmentosum variant phenotype. Here, we report experiments demonstrating that second human homolog (RAD30B), also a novel designate poliota. poliota, is distributive enzyme highly error-prone when replicating undamaged DNA. At template G or C, average error frequency was approximately...

Damage-induced SOS mutagenesis requiring the UmuD′C proteins occurs as part of cells’ global response to DNA damage. In vitro studies on biochemical basis have been hampered by difficulties in obtaining biologically active UmuC protein, which, when overproduced, is insoluble aqueous solution. We circumvented this problem purifying UmuD′ 2 C complex soluble form and used it reconstitute an lesion bypass system . Stimulated a site-directed model abasic presence C, activated RecA protein...

DNA polymerase iota (pol iota) is one of several recently discovered polymerases in mammalian cells whose function unknown. We report here that human pol has an intrinsic 5'-deoxyribose phosphate (dRP) lyase activity. In reactions reconstituted with uracil-DNA glycosylase (UDG), apurinic/apyrimidinic (AP) endonuclease and ligase I, can use its dRP activities to repair G*U A*U pairs DNA. These data three distinct catalytic properties implicate it specialized forms base excision (BER).

Recent studies suggest that DNA polymerase η (polη) and ι (polι) are involved in somatic hypermutation of immunoglobulin variable genes. To test the role polι generating mutations an animal model, we first characterized biochemical properties murine polι. Like its human counterpart, is extremely error-prone when catalyzing synthesis on a variety templates vitro. Interestingly, filling 1 base-pair gap, subsequent strand displacement was greatest presence both pols η. Genomic sequence analysis...

DNA damage-inducible mutagenesis in Escherichia coli is largely dependent upon the activity of UmuD (UmuD') and UmuC proteins. The intracellular level these proteins tightly regulated at both transcriptional posttranslational levels. Such regulation presumably allows cells to deal with damage via error-free repair pathways before being committed error-prone pathways. We have recently discovered that as part this elaborate regulation, are rapidly degraded vivo. report here enzyme responsible...

Most SOS mutagenesis in Escherichia coli is dependent on the UmuD and UmuC proteins. Perhaps as a consequence, activity of these proteins exquisitely regulated. The intracellular level normally quite low but increases dramatically lon − strains, suggesting that both are substrates Lon protease. We report here highly purified protein specifically degraded vitro by an ATP-dependent manner. To identify regions necessary for Lon-mediated proteolysis, we performed ‘alanine-stretch’ umuD followed...

All DNA polymerases require a divalent cation for catalytic activity. It is generally assumed that Mg(2+) the physiological cofactor replicative in vivo. However, recent studies suggest certain repair polymerases, such as pol lambda, may preferentially utilize Mn(2+) vitro. Here we report on effects of and enzymatic properties human polymerase iota (pol iota). exhibited greatest activity presence low levels (0.05-0.25 mm). Peak was observed range 0.1-0.5 mm significantly reduced at...

In addition to its critical role in genetic recombination, the Escherichia coli RecA protein plays a pivotal SOS-induced mutagenesis. This can be separated genetically into three steps: (i) depression of SOS regulon by mediating posttranslational cleavage LexA repressor, (ii) activation UmuD'-like proteins UmuD-like proteins, and (iii) direct step, possibly interact with target Umu-like mutagenesis lesions DNA. We have analyzed RecA's third biochemically using affinity chromatography an...

N 3-methyl-adenine (3MeA) is the major cytotoxic lesion formed in DNA by S 2 methylating agents. The presumably blocks progression of cellular replicases because 3-methyl group hinders interactions between polymerase and minor groove DNA. However, this hypothesis has yet to be rigorously proven, as 3MeA intrinsically unstable converted an abasic site, which itself a blocking lesion. To circumvent these problems, we have chemically synthesized 3-deaza analog (3dMeA) stable phosphoramidite...

Human DNA polymerase ι (polι) is a Y‐family whose cellular function presently unknown. Here, we report on the ability of polι to bypass various stereoisomers benzo[a]pyrene (BaP) diol epoxide (DE) and benzo[c]phenanthrene (BcPh) DE adducts at deoxyadenosine (dA) or deoxyguanosine (dG) bases in four different template sequence contexts vitro. We find that BaP dG pose strong block polι‐dependent replication result high frequency base misincorporations. In contrast, misincorporations opposite...

The activity of a number proteins is regulated by self-processing reactions. Elegant examples are the cleavage prokaryotic LexA and lambdaCI transcriptional repressors UmuD-like mutagenesis proteins. Various studies support hypothesis that reactions predominantly intramolecular in nature. recently described crystal structure Escherichia coli UmuD' protein (the posttranslational product UmuD protein) suggests, however, region corresponding to site at least 50 A away from catalytic active...

Several important anti-tumor agents form DNA interstrand crosslinks (ICLs), but their clinical efficiency is counteracted by multiple complex repair pathways. All of these pathways require unhooking the ICL from one strand a duplex nucleases, followed bypass unhooked translesion synthesis (TLS) polymerases. The structures ICLs remain unknown, yet position incisions and processing significantly influence fidelity TLS We have synthesized panel model nitrogen mustard to systematically...

The Escherichia coli Umu proteins play critical roles in damage-inducible SOS mutagenesis. To avoid any gratuitous mutagenesis, the activity of is normally kept to a minimum by tight transcriptional and posttranslational regulation. We have, however, previously observed that compared with an isogenic recA+ strain, steady-state levels are elevated recA730 background (R. Woodgate D. G. Ennis, Mol. Gen. Genet. 229:10-16, 1991). have investigated this phenomenon further find another...

Background Damage induced 'SOS mutagenesis' may occur transiently as part of the global SOS response to DNA damage in bacteria. A key participant this process is UmuD protein, which produced an inactive form but converted active form, UmuD′, by a RecA-mediated self-cleavage reaction. together with UmuC and activated RecA (RecA∗), enables polymerase III holoenzyme replicate across chemical UV lesions. The efficiency reaction depends on several intricate protein–protein interactions.Results...

α-OH-PdG, an acrolein-derived deoxyguanosine adduct, inhibits DNA synthesis and miscodes significantly in human cells. To probe the cellular mechanism underlying error-free error-prone translesion syntheses, vitro primer extension experiments using purified polymerases site-specific α-OH-PdG were conducted. The results suggest involvement of pol η synthesis. Experiments with xeroderma pigmentosum variant cells, which lack η, confirmed this hypothesis. also suggested ι and/or REV1 inserting...

DNA polymerase ι (Pol ι) is an attractive candidate for somatic hypermutation in antibody genes because of its low fidelity. To identify a role Pol ι, we analyzed mutations two strains mice with deficiencies the enzyme: 129 negligible expression truncated and knock-in that express full-length catalytically inactive. Both had normal frequencies spectra variable region, indicating loss did not change overall mutagenesis. We next examined if affected tandem generated by another error-prone...