The mutagenesis protein UmuC is a DNA polymerase activated by UmuD', RecA, and SSB and is specialized for translesion replication.
(25/1119)
Replication of DNA lesions leads to the formation of mutations. In Escherichia coli this process is regulated by the SOS stress response, and requires the mutagenesis proteins UmuC and UmuD'. Analysis of translesion replication using a recently reconstituted in vitro system (Reuven, N. B., Tomer, G., and Livneh, Z. (1998) Mol. Cell 2, 191-199) revealed that lesion bypass occurred with a UmuC fusion protein, UmuD', RecA, and SSB in the absence of added DNA polymerase. Further analysis revealed that UmuC was a DNA polymerase (E. coli DNA polymerase V), with a weak polymerizing activity. Upon addition of UmuD', RecA, and SSB, the UmuC DNA polymerase was greatly activated, and replicated a synthetic abasic site with great efficiency (45% bypass in 6 min), 10-100-fold higher than E. coli DNA polymerases I, II, or III holoenzyme. Analysis of bypass products revealed insertion of primarily dAMP (69%), and to a lesser degree dGMP (31%) opposite the abasic site. The UmuC104 mutant protein was defective both in lesion bypass and in DNA synthesis. These results indicate that UmuC is a UmuD'-, RecA-, and SSB-activated DNA polymerase, which is specialized for lesion bypass. UmuC is a member of a new family of DNA polymerases which are specialized for lesion bypass, and include the yeast RAD30 and the human XP-V genes, encoding DNA polymerase eta. (+info)
Multiple competition reactions for RPA order the assembly of the DNA polymerase delta holoenzyme.
(26/1119)
Processive extension of DNA in eukaryotes requires three factors to coordinate their actions. First, DNA polymerase alpha-primase synthesizes the primed site. Then replication factor C loads a proliferating cell nuclear antigen (PCNA) clamp onto the primer. Following this, DNA polymerase delta assembles with PCNA for processive extension. This report shows that these proteins each bind the primed site tightly and trade places in a highly coordinated fashion such that the primer terminus is never left free of protein. Replication protein A (RPA), the single-stranded DNA-binding protein, forms a common touchpoint for each of these proteins and they compete with one another for it. Thus these protein exchanges are driven by competition-based protein switches in which two proteins vie for contact with RPA. (+info)
The essential DNA polymerases delta and epsilon are involved in repair of UV-damaged DNA in the yeast Saccharomyces cerevisiae.
(27/1119)
We have studied the ability of yeast DNA polymerases to carry out repair of lesions caused by UV irradiation in Saccharomyces cerevisiae. By the analysis of postirradiation relative molecular mass changes in cellular DNA of different DNA polymerases mutant strains, it was established that mutations in DNA polymerases delta and epsilon showed accumulation of single-strand breaks indicating defective repair. Mutations in other DNA polymerase genes exhibited no defects in DNA repair. Thus, the data obtained suggest that DNA polymerases delta and epsilon are both necessary for DNA replication and for repair of lesions caused by UV irradiation. The results are discussed in the light of current concepts concerning the specificity of DNA polymerases in DNA repair. (+info)
Replisome assembly at oriC, the replication origin of E. coli, reveals an explanation for initiation sites outside an origin.
(28/1119)
This study outlines the events downstream of origin unwinding by DnaA, leading to assembly of two replication forks at the E. coli origin, oriC. We show that two hexamers of DnaB assemble onto the opposing strands of the resulting bubble, expanding it further, yet helicase action is not required. Primase cannot act until the helicases move 65 nucleotides or more. Once primers are formed, two molecules of the large DNA polymerase III holoenzyme machinery assemble into the bubble, forming two replication forks. Primer locations are heterogeneous; some are even outside oriC. This observation generalizes to many systems, prokaryotic and eukaryotic. Heterogeneous initiation sites are likely explained by primase functioning with a moving helicase target. (+info)
Double-strand-break repair recombination in Escherichia coli: physical evidence for a DNA replication mechanism in vivo.
(29/1119)
DNA double-strand-break repair (DSBR) is, in many organisms, accomplished by homologous recombination. In Escherichia coli DSBR was thought to result from breakage and reunion of parental DNA molecules, assisted by known endonucleases, the Holliday junction resolvases. Under special circumstances, for example, SOS induction, recombination forks were proposed to initiate replication. We provide physical evidence that this is a major alternative mechanism in which replication copies information from one chromosome to another generating recombinant chromosomes in normal cells in vivo. This alternative mechanism can occur independently of known Holliday junction cleaving proteins, requires DNA polymerase III, and produces recombined DNA molecules that carry newly replicated DNA. The replicational mechanism underlies about half the recombination of linear DNA in E. coli; the other half occurs by breakage and reunion, which we show requires resolvases, and is replication-independent. The data also indicate that accumulation of recombination intermediates promotes replication dramatically. (+info)
Strand asymmetry of +1 frameshift mutagenesis at a homopolymeric run by DNA polymerase III holoenzyme of Escherichia coli.
(30/1119)
We have recently shown that single-base frameshifts were predominant among mutations induced within the rpsL target sequence upon oriC plasmid DNA replication in vitro. We found that the occurrence of +1 frameshifts at a run of 6 residues of dA/dT could be increased proportionally by increasing the concentration of dATP present in the in vitro replication. Using single-stranded circular DNA containing either the coding sequence of the rpsL gene or its complementary sequence, the +1 frameshift mutagenesis by DNA polymerase III holoenzyme of Escherichia coli was extensively examined. A(6) --> A(7) frameshifts occurred 30 to 90 times more frequently during DNA synthesis with the noncoding sequence (dT tract) template than with the coding sequence (dA tract). Excess dATP enhanced the occurrence of +1 frameshifts during DNA synthesis with the dT tract template, but no other dNTPs showed such an effect. In the presence of 0.1 mM dATP, the A(6) --> A(7) mutagenesis with the dT tract template was not inhibited by 1.5 mM dCTP, which is complementary to the residue immediately upstream of the dT tract. These results strongly suggested that the A(6) --> A(7) frameshift mutagenesis possesses an asymmetric strand nature and that slippage errors leading to the +1 frameshift are made during chain elongation within the tract rather than by misincorporation of nucleotides opposite residues next to the tract. (+info)
Long patch base excision repair with purified human proteins. DNA ligase I as patch size mediator for DNA polymerases delta and epsilon.
(31/1119)
Among the different base excision repair pathways known, the long patch base excision repair of apurinic/apyrimidinic sites is an important mechanism that requires proliferating cell nuclear antigen. We have reconstituted this pathway using purified human proteins. Our data indicated that efficient repair is dependent on six components including AP endonuclease, replication factor C, proliferating cell nuclear antigen, DNA polymerases delta or epsilon, flap endonuclease 1, and DNA ligase I. Fine mapping of the nucleotide replacement events showed that repair patches extended up to a maximum of 10 nucleotides 3' to the lesion. However, almost 70% of the repair synthesis was confined to 2-4-nucleotide patches and DNA ligase I appeared to be responsible for limiting the repair patch length. Moreover, both proliferating cell nuclear antigen and flap endonuclease 1 are required for the production and ligation of long patch repair intermediates suggesting an important role of this complex in both excision and resynthesis steps. (+info)
9-(2-phosphonylmethoxyethyl) derivatives of purine nucleotide analogs: A comparison of their metabolism and interaction with cellular DNA synthesis.
(32/1119)
Incubation of CEM cells for 24 h with the guanine, 2,6-diaminopurine, and adenine nucleotide analogs of the 9-(2-phosphonylmethoxyethyl) series, 9-(2-phosphonylmethoxyethyl)guanine (PMEG), 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP), and 9-(2-phosphonylmethoxyethyl)adenine (PMEA), was found to inhibit DNA synthesis 50% at concentrations of 1, 6, and 25 microM, respectively. Possible reasons for the marked differences were investigated, including cellular transport of the analogs, different efficiencies of intracellular phosphorylation, differential effects on 2'-deoxynucleotide (dNTP) pools, and differences in the affinities of the cellular DNA polymerases for the diphosphate derivatives of the drugs. No significant differences in cellular uptake were found among the analogs; however, they did differ in the efficiency of phosphorylation, i.e., CEM cells were found to accumulate higher levels of PMEG-diphosphate (PMEGpp) than PMEDAP-diphosphate (PMEDAPpp) or PMEA-diphosphate (PMEApp). Treatment of cells with any of the nucleotide analogs resulted in increased dNTP pools, with PMEG producing the greatest increase. All three analogs had the greatest effect on the dATP pool size, whereas the dGTP pool size was not significantly affected. Comparison of the ratios of nucleotide analog diphosphates to their corresponding dNTPs under conditions where DNA synthesis is inhibited 50% suggested that cellular DNA polymerases were approximately twice as sensitive to PMEGpp than to PMEDAPpp and 5-fold more sensitive to PMEGpp than to PMEApp. Consistent with this hypothesis, examination of the efficiencies with which the replicative DNA polymerases alpha, delta, and epsilon incorporated the analogs showed that DNA polymerase delta, the most sensitive of the DNA polymerases, incorporated PMEGpp twice as efficiently as PMEDAPpp and 7-fold more efficiently than PMEApp. (+info)