The dinB gene encodes a novel E. coli DNA polymerase, DNA pol IV, involved in mutagenesis.

In Escherichia coli, the dinB gene is required for the SOS-induced lambda untargeted mutagenesis pathway and confers a mutator phenotype to the cell when the gene product is overexpressed. Here, we report that the purified DinB protein is a DNA polymerase. This novel E. coli DNA polymerase (pol IV) is shown to be strictly distributive, devoid of proofreading activity, and prone to elongate bulged (misaligned) primer/template structures. Site-directed mutagenesis experiments of dinB also demonstrate that the polymerase activity of DinB is required for its in vivo mutagenicity. Along with the sequence homologies previously found within the UmuC-like protein family, these results indicate that the uncovered DNA polymerase activity may be a common feature of all these homologous proteins.     

DNA replication through G-quadruplex motifs is promoted by the Saccharomyces cerevisiae Pif1 DNA helicase

G-quadruplex (G4) DNA structures are extremely stable four-stranded secondary structures held together by noncanonical G-G base pairs. Genome-wide chromatin immunoprecipitation was used to determine the in?vivo binding sites of the multifunctional Saccharomyces cerevisiae Pif1 DNA helicase, a potent unwinder of G4 structures in?vitro. G4 motifs were a significant subset of the high-confidence Pif1-binding sites. Replication slowed in the vicinity of these motifs, and they were prone to breakage in Pif1-deficient cells, whereas non-G4 Pif1-binding sites did not show this behavior. Introducing many copies of G4 motifs caused slow growth in replication-stressed Pif1-deficient cells, which was relieved by spontaneous mutations that eliminated their ability to form G4 structures, bind Pif1, slow DNA replication, and stimulate DNA breakage. These data suggest that G4 structures form in?vivo and that they are resolved by Pif1 to prevent replication fork stalling and DNA breakage.     

Isolation of a yeast single-strand deoxyribonucleic acid binding protein that specifically stimulates yeast DNA polymerase I

We sought a protein from yeast that would bind more strongly to single-stranded DNA than to duplex DNA and would stimulate the activity of the major yeast DNA polymerase, but not polymerases from other organisms. We isolated a protein that binds about 200 times more strongly to single-stranded DNA than duplex DNA and stimulates yeast DNA polymerase I activity 4-5-fold. It inhibits synthesis catalyzed by calf thymus DNA polymerase alpha and has little effect on T4 DNA polymerase. This yeast protein, SSB-1, has a molecular weight of approximately 40 000. At apparent saturation there is one protein molecule bound per 40 nucleotides. Protein binding causes the single-stranded DNA molecule to assume a relatively extended conformation. It binds to single-stranded RNA as strongly as to DNA. SSB-1 increases the initial rate of polymerization catalyzed by yeast DNA polymerase I apparently by increasing the processivity of the enzyme. We estimate there are 7500-30 000 molecules of SSB-1 per yeast cell, enough to bind at least 400-1600 nucleotides per replication fork. Thus it is present in sufficient abundance to participate in DNA replication in vivo in the manner suggested by these in vitro experiments.     

The effect of epinephrine and serotonin on hepatic poly(A)+ RNA synthesis

In vivo administration of epinephrine or serotonin has been shown to stimulate the incorporation of 14C-orotic acid into Poly(A)+ RNA. However, only epinephrine and not serotonin could stimulate DNA dependent RNA polymerase activity of isolated hepatic nuclei in in vitro experiments.     

Slipped Misalignment Mechanisms of Deletion Formation: In Vivo Susceptibility to Nucleases

Misalignment of repeated sequences during DNA replication can lead to deletions or duplications in genomic DNA. In Escherichia coli, such genetic rearrangements can occur at high frequencies, independent of the RecA-homologous recombination protein, and are sometimes associated with sister chromosome exchange (SCE). Two mechanisms for RecA-independent genetic rearrangements have been proposed: simple replication misalignment of the nascent strand and its template and SCE-associated misalignment involving both nascent strands. We examined the influence of the 3′ exonuclease of DNA polymerase III and exonuclease I on deletion via these mechanisms in vivo. Because mutations in these exonucleases stimulate tandem repeat deletion, we conclude that displaced 3′ ends are a common intermediate in both mechanisms of slipped misalignments. Our results also confirm the notion that two distinct mechanisms contribute to slipped misalignments: simple replication misalignment events are sensitive to DNA polymerase III exonuclease, whereas SCE-associated events are sensitive to exonuclease I. If heterologies are present between repeated sequences, the mismatch repair system dependent on MutS and MutH aborts potential deletion events via both mechanisms. Our results suggest that simple slipped misalignment and SCE-associated misalignment intermediates are similarly susceptible to destruction by the mismatch repair system.     

Protein sequences conserved in prokaryotic aminoacyl-tRNA synthetases are important for the activity of the processivity factor of human mitochondrial DNA polymerase

Previous studies have shown that the small subunit of Xenopus DNA polymerase gamma (pol gammaB) acts as a processivity factor to stimulate the 140 kDa catalytic subunit of human DNA polymerase gamma. A putative human pol gammaB initially identified by analysis of DNA sequence had not been shown to be functional, and appeared to be an incomplete clone. In this paper, we report the cloning of full-length human and mouse pol gammaB. Both human and mouse pol gammaB proteins were expressed in their mature forms, without their apparent mitochondrial localization signals, and shown to stimulate processivity of the recombinant catalytic subunit of human pol gammaA. Deletion analysis of human pol gammaB indicated that blocks of sequence conserved with prokaryotic class II aminoacyl-tRNA synthetases are necessary for activity and inter-action with human pol gammaA. Purification of DNA pol gamma from HeLa cells indicated that both proteins are associated in vivo.     

Escherichia coli DNA polymerase II is stimulated by DNA polymerase III holoenzyme auxiliary subunits

DNA polymerase III of Escherichia coli requires multiple auxiliary factors to enable it to serve as a replicative complex. We demonstrate that auxiliary components of the DNA polymerase III holoenzyme, the gamma delta complex and beta subunit, markedly stimulate DNA polymerase II on long single-stranded templates. DNA polymerase II activity is enhanced by single-stranded DNA binding protein, but the stimulation by gamma delta and beta can be observed either in the absence or presence of single-stranded DNA binding protein. In contrast with DNA polymerase III, the requirement of DNA polymerase II for gamma delta cannot be bypassed by large excesses of the beta subunit at low ionic strength in the absence of the single-stranded DNA binding protein. The product of the DNA polymerase II-gamma delta-beta reaction on a uniquely primed single-stranded circle is of full template length; the reconstituted enzyme apparently is incapable of strand displacement synthesis. The possible biological implications of these observations are discussed.     

Relationship between poly(ADP-ribose) polymerase activity and DNA synthesis in cultured hepatocytes

Previous studies have demonstrated that an increase in poly(ADP-ribose) polymerase activity could be closely related to DNA replication during liver regeneration and to DNA repair synthesis in different experimental systems. This relationship was further investigated by studying the time course of endogenous and total poly(ADP-ribose) polymerase activity in cultured rat hepatocytes stimulated by epidermal growth factor. This mitogen has been shown to stimulate DNA synthesis in liver cells both in vivo and in vitro. A 6-fold increase in endogenous activity was observed early after epidermal growth factor addition, just before DNA synthesis. A subsequent 4-fold increment in total enzyme activity, concomitant with DNA synthesis, was detected. Orotic acid, which has recently shown mitoinhibitory effect, abolished the epidermal-growth-factor-induced increase in endogenous and total poly(ADP-ribose) polymerase activity, as well as DNA synthesis. On the contrary, 3-aminobenzamide inhibitor of poly(ADP-ribose) polymerase completely suppressed the endogenous activity but only partially modified the increase in total catalytic level and the overall pattern of thymidine incorporation. Taken together, these data indicate that, in cultured hepatocytes, the induction of DNA synthesis is supported by an increased poly(ADP-ribose) polymerase activity.     

Necessity of polyamines for maximum in vivo synthesis of beta beta' subunits of RNA polymerase

The possibility that polyamines can stimulate the in vivo synthesis of beta beta' subunits of RNA polymerase has been examined through the use of a polyamine-requiring mutant of Escherichia coli. Results from autoradiographic estimation and activity measurement of RNA polymerase in cell extracts prepared from polyamine starved and unstarved bacteria showed that polyamines stimulate the synthesis of beta beta' subunits of RNA polymerase 2- to 3.6-fold.     

Reduced stimulation of recombinant DNA polymerase γ and mitochondrial DNA (mtDNA) helicase by variants of mitochondrial single-stranded DNA-binding protein (mtSSB) correlates with defects in mtDNA replication in animal cells

The mitochondrial single-stranded DNA-binding protein (mtSSB) is believed to coordinate the functions of DNA polymerase γ (pol γ) and the mitochondrial DNA (mtDNA) helicase at the mtDNA replication fork. We generated five variants of the human mtSSB bearing mutations in amino acid residues specific to metazoans that map on the protein surface, removed from the single-stranded DNA (ssDNA) binding groove. Although the mtSSB variants bound ssDNA with only slightly different affinities, they exhibited distinct capacities to stimulate the DNA polymerase activity of human pol γ and the DNA unwinding activity of human mtDNA helicase in vitro. Interestingly, we observed that the variants with defects in stimulating pol γ had unaltered capacities to stimulate the mtDNA helicase; at the same time, variants showing reduced stimulation of the mtDNA helicase activity promoted DNA synthesis by pol γ similarly to the wild-type mtSSB. The overexpression of the equivalent variants of Drosophila melanogaster mtSSB in S2 cells in culture caused mtDNA depletion under conditions of mitochondrial homeostasis. Furthermore, we observed more severe reduction of mtDNA copy number upon expression of these proteins during recovery from treatment with ethidium bromide, when mtDNA replication is stimulated in vivo. Our findings suggest that mtSSB uses distinct structural elements to interact functionally with its mtDNA replisome partners and to promote proper mtDNA replication in animal cells.     

Single-stranded DNA structure and DNA polymerase activity in the presence of nucleic acid helix-unwinding proteins from calf thymus.

In the preceding articles we have described the isolation and some of the properties of two calf thymus proteins which bind selectively to single-stranded DNA and which appear analogous to previously isolated prokaryotic DNA-unwinding proteins. In the present work we demonstrate two further points of analogy. First, both the calf UP1 and the high salt eluting proteins form protein-rich complexes with single-stranded DNA, and hold this DNA in a rigid, extended conformation. Second, these proteins stimulate the calf thymus DNA polymerase-alpha; phage T4 gene 32-protein does not. The stimulation of a homologous DNA polymerase is characteristic of several prokaryotic DNA-unwinding proteins and is assumed to reflect their in vivo role in DNA synthesis.     

Regulatory roles of p21 and apurinic/apyrimidinic endonuclease 1 in base excision repair

Many types of DNA damage induce a cellular response that inhibits replication but allows repair by up-regulating the p53 pathway and inducing p21(Cip1, Waf1, Sdi1). The p21 regulatory protein can bind proliferating cell nuclear antigen (PCNA) and prohibit DNA replication. We show here that p21 also inhibits PCNA stimulation of long patch base excision repair (BER) in vitro. p21 disrupts PCNA-directed stimulation of flap endonuclease 1 (FEN1), DNA ligase I, and DNA polymerase delta. The dilemma is to understand how p21 prevents DNA replication but allows BER in vivo. Differential regulation by p21 is likely to relate to the utilization of DNA polymerase beta, which is not sensitive to p21, in the repair pathway. We have also found that apurinic/apyrimidinic endonuclease 1 (APE1) stimulates long patch BER. Furthermore, neither APE1 activity nor its ability to stimulate long patch BER is significantly affected by p21 in vitro. We propose that APE1 serves as an assembly and coordination factor for long patch BER proteins. APE1 initially cleaves the DNA and then facilitates the sequential binding and catalysis by DNA polymerase beta, DNA polymerase delta, FEN1, and DNA ligase I. This model implies that BER can be regulated differentially, based upon the assembly of relevant proteins around APE1 in the presence or absence of PCNA.     

Coordination between the polymerase and 5'-nuclease components of DNA polymerase I of Escherichia coli

The polymerase and 5'-nuclease components of DNA polymerase I must collaborate in vivo so as to generate ligatable structures. Footprinting shows that the polymerase and 5'-nuclease cannot bind simultaneously to a DNA substrate and appear to compete with one another, suggesting that the two active sites are physically separate and operate independently. The desired biological end point, a ligatable nick, results from the substrate specificities of the polymerase and 5'-nuclease. The preferred substrate of the 5'-nuclease is a "double-flap" structure having a frayed base at the primer terminus overlapping the displaced strand that is to be cleaved by the 5'-nuclease. Cleavage of this structure occurs almost exclusively between the first two paired bases of the downstream strand, yielding a ligatable nick. In whole DNA polymerase I, the polymerase and 5'-nuclease activities are coupled such that the majority of molecules cleaved by the 5'-nuclease have also undergone polymerase-catalyzed addition to the primer terminus. This implies that the 5'-nuclease can capture a DNA molecule from the polymerase site more efficiently than from the bulk solution.     

Phosphorylation of DNA polymerase.

Both E. coli and calf thymus DNA polymerase can be phosphorylated by cAMP-dependent protein kinase and phosphorylation appears to stimulate the DNA polymerase reaction. Conversely, dephosphorylation of the polymerase molecule, by a protein phosphatase, inhibits the polymerase reaction.     

Mispair formation in DNA can involve rare tautomeric forms in the template.

The formation of pyridine-pyrimidine- and pyrimidine-pyrimidine base pairs after in vitro DNA replication with the large fragment of Escherichia coli DNA polymerase I indicates that Watson-Crick-like base pairing between pyrimidine bases can occur in the enzyme due to the presence of the rare tautomers of deoxycytidylate and thymidylate in the template strand. The implications to mispair formation in DNA, such as the difference between the structures of the mispairs during and after replication, are discussed and the possible action of mutagenic DNA protonating and deprotonating agents in vivo is considered.