Saccharomyces cerevisiae possesses two functional homologues (Ntg1p and Ntg2p) of the Escherichia coli endonuclease III protein, a DNA base excision repair N-glycosylase with a broad substrate specificity directed primarily against oxidatively damaged pyrimidines. The substrate specificities of Ntg1p and Ntg2p are similar but not identical, and differences in their amino acid sequences as well as inducibility by DNA damaging agents suggest that the two proteins may have different biological roles and subcellular locations. Experiments performed on oligonucleotides containing a variety of oxidative base damages indicated that dihydrothymine, urea, and uracil glycol are substrates for Ntg1p and Ntg2p, although dihydrothymine was a poor substrate for Ntg2p. Vectors encoding Ntg1p-green fluorescent protein (GFP) and Ntg2p-GFP fusions under the control of their respective endogenous promoters were utilized to observe the subcellular targeting of Ntg1p and Ntg2p in S. cerevisiae. Fluorescence microscopy of pNTG1-GFP and pNTG2-GFP transformants revealed that Ntg1p localizes primarily to the mitochondria with some nuclear localization, whereas Ntg2p localizes exclusively to the nucleus. In addition, the subcellular location of Ntg1p and Ntg2p confers differential sensitivities to the alkylating agent MMS. These results expand the known substrate specificities of Ntg1p and Ntg2p, indicating that their base damage recognition ranges show distinct differences and that these proteins mediate different roles in the repair of DNA base damage in the nucleus and mitochondria of yeast.

There is increasing evidence that four-stranded Hoogsteen-bonded DNA structures, G4-DNA, play an important role in cellular processes such as meiosis and recombination. The Hoogsteen-bonded G4-DNA is thermodynamically more stable than duplex DNA, and many guanine-rich genomic DNA sequences with the ability to form G4-DNA have been identified. A protein-dependent activity that resolves G4-DNA into single-stranded DNA has been identified in human placental tissue. The resolvase activity was purified from any apparent nuclease activity and is dependent on NTP hydrolysis and MgCl 2 . Resolvase activity is optimal with 5 mM MgCl 2 . The V max /K m of ATP is 0.055%/min/ M, higher than the V max /K m of the other dNTPs. The products of the resolvase reaction are unmodified single-stranded DNA. The resolvase is not a duplex DNA helicase or a topoisomerase II activity and does not unwind Hoogsteenbonded triplex DNA. Resolvase is a novel activity that unwinds stable G4-DNA structures using a dNTPdependent mechanism producing unmodified singlestranded DNA. Potential in vivo roles for this G4-DNA resolvase activity are discussed.Guanine-rich DNA sequences that form G4-DNA are found in a number of evolutionarily conserved genomic regions such as telomeres, dimerization domains of retroviruses, and the insulin gene promoter (1-5). The four-stranded structure requires a monovalent cation to form, and the DNA strands can run in either a parallel or anti-parallel orientation (6 -8). G4-DNA contains Hoogsteen bonds between the guanine residues forming square planar guanine quartets (9). X-ray crystal diffraction and two-dimensional nuclear magnetic resonance show the sugar backbone can exist in many variations (10 -12). Guanine quartets have unusual stacking energy and high stability. The ability of the O-6 of guanine to form a coordination complex with either Na ϩ or K ϩ in guanine quartets is thought to stabilize telomeres (6 -8). The thermodynamic parameters of parrellel-stranded G4-DNA are indicative of its stability with the free energy of formation equal to Ϫ21 kcal/mol and the transition temperature above 82°C (13). DNA sequences able to form G4-DNA have also been found at sites of spontaneous gene rearrangements, point mutations and, along with triplex DNA, have been implicated in causing DNA mutations (9, 14 -16).Many different proteins with specificity for binding to G4-DNA have been identified (17)(18)(19)(20)(21)(22). The identification of a G4-DNA-specific nuclease from yeast as the SEP1/KEM1 protein, and the meiotic block at the 4N stage for KEM1-null cells, supports the hypothesis that G4-DNA is involved in meiosis (23, 24, 9). More recently, two yeast gene products with specific activity for G4-DNA were cloned and sequenced, G4p1 and G4p2 (19, 20). G4p1 is a homodimer of the gene encoding a novel protein with a domain homologous to the bacterial methionyl-tRNA synthetase dimerization domains, and G4p2 is encoded by a gene identical to genes that appear to function in protein kinase-controlled signal transduction,...

Oligonucleotides containing a specific initiation site for polymerase ␣-primase (pol ␣-primase) were used to measure the effects of cytosine arabinoside triphosphate and cytosine arabinoside monophosphate (araCMP) in DNA on RNA-primed DNA synthesis. Primase inserts araCMP at the 3 terminus of a full-length RNA primer with a 400-fold preference over CMP. The araCMP is elongated efficiently by pol ␣ in the primasecoupled reaction. Extension from RNA 3-araCMP is 50-fold less efficient than from CMP, and extension from DNA 3-araCMP is 1600-fold less efficient than from dCMP. Using araCMP-containing templates, primer synthesis is reduced 2-3-fold, and RNA-primed DNA synthesis is reduced 2-8-fold. The efficiency of polymerization past a template araCMP by pol ␣ is reduced 180-fold during insertion of dGMP opposite araCMP and 35-fold during extension from the araCMP:dGMP 3 terminus. These results show that the pol ␣-primase efficiently incorporates araCMP as the border nucleotide between RNA and DNA and suggest that the inhibitory effects of araC most likely result from slowed elongation of pol ␣ and less so from inhibition of primer synthesis by primase.In cells, RNA-primed DNA fragments are synthesized at origins of replication and on lagging strands at replication forks (1, 2). Initiation of DNA chains requires oligoribonucleotide synthesis to provide 3Ј-hydroxyl termini for DNA polymerase elongation. The tight association between DNA polymerase ␣ (pol ␣) 1 and primase indicate that pol ␣-primase complex is responsible for RNA-primed DNA synthesis. Primase synthesizes RNA primers that are about 10 Nts in length de novo, and pol ␣ elongates the primers.The aranucleotides inhibit DNA replication in animal cells (3). Inhibition by the triphosphate forms of these analogues might result during RNA-primed DNA synthesis by pol ␣-primase (4). Incorporation into RNA primers by primase and into DNA by pol ␣ is at positions opposite complementary nucleotides (3-10). Primase inserts araATP and FaraATP more efficiently than ATP (9, 11), and pol ␣ inserts these analogues as efficiently as deoxynucleotides (12). Insertion of aranucleotides terminates primer chain elongation by primase (9, 11), and 3Ј-aranucleotides in DNA decrease elongation by pol ␣ 2000-fold (12). In contrast, aranucleotides at RNA 3Ј termini only moderately decrease elongation by pol ␣ (9, 11). The synthesis of nonfunctional primers less than 7 Nts in length using homopolymer templates has made it difficult to quantitate effects of aranucleotides during RNA-primed DNA synthesis and to distinguish effects on primase and pol ␣.Oligonucleotide templates permit analysis of sequence-specific effects of aranucleotides on RNA-primed DNA synthesis. The sequences of some primer initiation sites have been identified (13-16), and these sequences support initiation in oligomer templates (15, 16). Using an oligonucleotide containing an initiation site for pol ␣-primase, primers with the sequence 5Ј-GGAAGAAAGC-3Ј are generated (16). Incorporation of araCMP at the 3Ј terminus...

The initiation of new DNA strands at origins of replication in animal cells requires de novo synthesis of RNA primers by primase and subsequent elongation from RNA primers by DNA polymerase alpha. To study the specificity of primer site selection by the DNA polymerase alpha-primase complex (pol alpha-primase), a natural DNA template containing a site for replication initiation was constructed. Two single-stranded DNA (ssDNA) molecules were hybridized to each other generating a duplex DNA molecule with an open helix replication 'bubble' to serve as an initiation zone. Pol alpha-primase recognizes the open helix region and initiates RNA-primed DNA synthesis at four specific sites that are rich in pyrimidine nucleotides. The priming site positioned nearest the ssDNA-dsDNA junction in the replication 'bubble' template is the preferred site for initiation. Using a 40 base oligonucleotide template containing the sequence of the preferred priming site, primase synthesizes RNA primers of 9 and 10 nt in length with the sequence 5'-(G)GAAGAAAGC-3'. These studies demonstrate that pol alpha-primase selects specific nucleotide sequences for RNA primer formation and suggest that the open helix structure of the replication 'bubble' directs pol alpha-primase to initiate RNA primer synthesis near the ssDNA-dsDNA junction.

The critical role of NTP concentration in determining where calf thymus DNA primase synthesizes a primer on a DNA template was examined. Varying the concentration of NTPs dramatically altered the template sequences at which primase synthesized primers. At the low NTP concentrations typically used for in vitro experiments (100 microM), primase greatly preferred to synthesize primers at pyrimidine rich DNA sequences. However, when the concentrations of NTPs were increased to levels typically found in whole cells, primers were now synthesized in all regions of the template. Importantly, synthesis of primers in all regions of the DNA template, not just the pyrimidine rich sequences, is the pattern of primer synthesis observed during DNA replication in whole cells. With low concentrations of NTPs (i.e., Vmax/K(M) conditions), primers are only synthesized at the most preferred synthesis sites, namely, those that are pyrimidine rich. In contrast, under conditions of high NTP concentrations, primer synthesis will occur at the first potential synthesis site to which primase binds. Now, the primase x DNA complex will be immediately converted to a primase x DNA x NTP x NTP complex that is poised for primer synthesis.

No abstract

No abstract