RESUMO
hHR23B was originally isolated as a component of a protein complex that specifically complements nucleotide excision repair (NER) defects of xeroderma pigmentosum group C cell extracts in vitro and was identified as one of two human homologs of the Saccharomyces cerevisiae NER gene product Rad23. Recombinant hHR23B has previously been shown to significantly stimulate the NER activity of recombinant human XPC protein (rhXPC). In this study we identify and functionally characterize the XPC-binding domain of hHR23B protein. We prepared various internal as well as terminal deletion products of hHR23B protein in a His-tagged form and examined their binding with rhXPC by using nickel-chelating Sepharose. We demonstrate that a domain covering 56 amino acids of hHR23B is required for binding to rhXPC as well as for stimulation of in vitro NER reactions. Interestingly, a small polypeptide corresponding to the XPC-binding domain is sufficient to exert stimulation of XPC NER activity. Comparison with known crystal structures and analysis with secondary structure programs provided strong indications that the binding domain has a predominantly amphipathic alpha-helical character, consistent with evidence that the affinity with XPC is based on hydrophobic interactions. Our work shows that binding to XPC alone is required and sufficient for the role of hHR23B in in vitro NER but does not rule out the possibility that the protein has additional functions in vivo.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Xeroderma Pigmentoso/metabolismo , Sequência de Aminoácidos , Especificidade de Anticorpos , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Reparo do DNA , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA/genética , Humanos , Imunoquímica , Técnicas In Vitro , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligonucleotídeos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Ubiquitinas/genética , Xeroderma Pigmentoso/genéticaRESUMO
A protein complex which specifically complements defects of XP-C cell extracts in vitro was previously purified to near homogeneity from HeLa cells. The complex consists of two tightly associated proteins: the XPC gene product and HHR23B, one of two human homologs of the Saccharomyces cerevisiae repair gene product Rad23 (Masutani et al., EMBO J. 13:1831-1843, 1994). To elucidate the roles of these proteins in "genome-overall" repair, we expressed the XPC protein in a baculovirus system and purified it to near homogeneity. The recombinant human XPC (rhXPC) protein exhibited a high level of affinity for single-stranded DNA and corrected the repair defect in XP-C whole-cell extracts without extra addition of recombinant HHR23B (rHHR23B) protein. However, Western blot (immunoblot) experiments revealed that XP-C cell extracts contained excess endogenous HHR23B protein, which might be able to form a complex upon addition of the rhXPC protein. To investigate the role of HHR23B, we fractionated the XP-C cell extracts and constructed a reconstituted system in which neither endogenous XPC nor HHR23B proteins were present. In this assay system, rhXPC alone weakly corrected the repair defect, while significant enhancement of the correcting activity was observed upon coaddition of rHHR23B protein. Stimulation of XPC by HHR23B was found with simian virus 40 minichromosomes as well as with naked plasmid DNA and with UV- as well as N-acetoxy-2- acetylfluorene-induced DNA lesions, indicating a general role of HHR23B in XPC functioning in the genome-overall nucleotide excision repair subpathway.
Assuntos
Reparo do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/fisiologia , Animais , Sequência de Bases , Enzimas Reparadoras do DNA , DNA Recombinante/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Nucleopoliedrovírus/genética , Proteínas Recombinantes de Fusão/metabolismo , Vírus 40 dos Símios/genética , Spodoptera , Xeroderma Pigmentoso/patologiaRESUMO
XPC-hHR23B protein complex is specifically involved in nucleotide excision repair (NER) of DNA lesions on transcriptionally inactive sequences as well as the nontranscribed strand of active genes. Here we demonstrate that not only highly purified recombinant hHR23B (rhHR23B) but also a second human homolog of the Saccharomyces cerevisiae Rad23 repair protein, hHR23A, stimulates the in vitro repair activity of recombinant human XPC (rhXPC), revealing functional redundancy between these human Rad23 homologs. Coprecipitation experiments with His-tagged rhHR23 as well as sedimentation velocity analysis showed that both rhHR23 proteins in vitro reconstitute a physical complex with rhXPC. Both complexes were more active than free rhXPC, indicating that complex assembly is required for the stimulation. rhHR23B was shown to stimulate an early stage of NER at or prior to incision. Furthermore, both rhHR23 proteins function in a defined NER system reconstituted with purified proteins, indicating direct involvement of hHR23 proteins in the DNA repair reaction via interaction with XPC.
Assuntos
Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Técnicas In Vitro , Substâncias Macromoleculares , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Frações Subcelulares/metabolismo , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/metabolismoRESUMO
Defects in the human gene XPV result in the variant form of the genetic disease xeroderma pigmentosum (XP-V). XPV encodes DNA polymerase eta, a novel DNA polymerase that belongs to the UmuC/DinB/Rad30 superfamily. This polymerase catalyzes the efficient and accurate translesion synthesis of DNA past cis-syn cyclobutane di-thymine lesions. In this report we present the cDNA sequence and expression profiles of the mouse XPV gene and demonstrate its ability to complement defective DNA synthesis in XP-V cells. The mouse XPV protein shares 80.3% amino acid identity and 86.9% similarity with the human XPV protein. The recombinant mouse XPV protein corrected the inability of XP-V cell extracts to carry out DNA replication, by bypassing thymine dimers on template DNA. Transfection of the mouse or human XPV cDNA into human XP-V cells corrected UV sensitivity. Northern blot analysis revealed that the mouse XPV gene is expressed ubiquitously, but at a higher level in testis, liver, skin and thymus compared to other tissues. Although the mouse XPV gene was not induced by UV irradiation, its expression was elevated approximately 4-fold during cell proliferation. These results suggest that DNA polymerase eta plays a role in DNA replication, though the enzyme is not essential for viability.
Assuntos
DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Teste de Complementação Genética , Mutação/genética , Tolerância a Radiação , Xeroderma Pigmentoso/enzimologia , Sequência de Aminoácidos , Animais , Divisão Celular , Linhagem Celular , Sobrevivência Celular/efeitos da radiação , Clonagem Molecular , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/química , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Variação Genética/genética , Humanos , Camundongos , Dados de Sequência Molecular , Dímeros de Pirimidina/genética , Dímeros de Pirimidina/metabolismo , Dímeros de Pirimidina/efeitos da radiação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tolerância a Radiação/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transfecção , Raios Ultravioleta , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/metabolismo , Xeroderma Pigmentoso/patologia , DNA Polimerase iotaRESUMO
The xeroderma pigmentosum variant (XP-V) is one of the most common forms of this cancer-prone syndrome. XP groups A through G are characterized by defective nucleotide excision repair, whereas the XP-V phenotype is proficient in this pathway. The XPV gene encodes DNA polymerase eta, which catalyzes an accurate translesion synthesis, indicating that the XPV gene contributes tumor suppression in normal individuals. Here we describe the genomic structure and chromosomal localization of the XPV gene, which includes 11 exons covering the entire coding sequence, lacks a TATA sequence in the upstream region of the transcription-initiation, and is located at the chromosome band 6p21.1-6p12. Analyses of patient-derived XP-V cell lines strongly suggested that three of four cell lines carried homozygous mutations in the XPV gene. The fourth cell line, XP1RO, carried heterozygous point mutations in the XPV gene, one of which was located at the splice acceptor site of exon 2, resulting in the omission of exon 2 from the mature mRNA. These findings provide a basis for diagnosis and therapy of XP-V patients.
Assuntos
Cromossomos Humanos Par 6/genética , DNA Polimerase Dirigida por DNA/genética , Genes , Xeroderma Pigmentoso/genética , Alelos , Animais , Sequência de Bases , Linhagem Celular , Mapeamento Cromossômico , Cromossomos Artificiais Humanos , Análise Mutacional de DNA , Éxons/genética , Células HeLa , Humanos , Células Híbridas , Hibridização in Situ Fluorescente , Íntrons/genética , Camundongos , Dados de Sequência Molecular , Transcrição Gênica , Xeroderma Pigmentoso/patologiaRESUMO
We have previously described the purification of an ultraviolet light (UV) damage-specific DNA-binding protein from Drosophila melanogaster, designated D-DDB P1 [Nucleic Acids Res., 23 (1995) 2600-2607]. Here, we obtained highly purified D-DDB P1 from Drosophila Kc cells, and we found that D-DDB P1 is also a nuclease. D-DDB P1 can selectively bind to pyrimidine (6-4) pyrimidone photoproducts, and in the presence of Mg++, D-DDB P1 can catalyze an incision immediately on the 3' and 5' sides of the (6-4) photoproduct site.
Assuntos
Dano ao DNA , DNA Bacteriano/efeitos da radiação , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Endodesoxirribonucleases/isolamento & purificação , Raios Ultravioleta , Animais , Sequência de Bases , Reparo do DNA , Drosophila melanogaster/enzimologia , Dados de Sequência MolecularRESUMO
We describe here the error specificity of mammalian DNA polymerase eta (pol eta), an enzyme that performs translesion DNA synthesis and may participate in somatic hypermutation of immunoglobulin genes. Both mouse and human pol eta lack intrinsic proofreading exonuclease activity and both copy undamaged DNA inaccurately. Analysis of more than 1500 single-base substitutions by human pol eta indicates that error rates for all 12 mismatches are high and variable depending on the composition and symmetry of the mismatch and its location. pol eta also generates tandem base substitutions at an unprecedented rate, and kinetic analysis indicates that it extends a tandem double mismatch about as efficiently as other replicative enzymes extend single-base mismatches. This ability to use an aberrant primer terminus and the high rate of single and double-base substitutions support the idea that pol eta may forego strict shape complementarity in order to facilitate highly efficient lesion bypass. Relaxed discrimination is further indicated by pol eta infidelity for a wide variety of nucleotide deletion and addition errors. The nature and location of these errors suggest that some may be initiated by strand slippage, while others result from additional mechanisms.
Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Mutagênese , Animais , Pareamento Incorreto de Bases/genética , Sequência de Bases , Dano ao DNA/genética , Análise Mutacional de DNA , DNA Polimerase Dirigida por DNA/química , Mutação da Fase de Leitura/genética , Genes de Imunoglobulinas/genética , Humanos , Cinética , Óperon Lac/genética , Camundongos , Dados de Sequência Molecular , Mutagênese/genética , Mutação Puntual/genética , Deleção de Sequência/genética , Especificidade por Substrato , Moldes GenéticosRESUMO
The XPC-HR23B complex, a mammalian factor specifically involved in global genomic nucleotide excision repair (NER) has been shown to bind various forms of damaged DNA and initiate DNA repair in cell-free reactions. To characterize the binding specificity of this factor in more detail, a method based on immunoprecipitation was developed to assess the relative affinity of XPC-HR23B for defined lesions on DNA. Here we show that XPC-HR23B preferentially binds to UV-induced (6-4) photoproducts (6-4PPs) as well as to cholesterol, but not to the cyclobutane pyrimidine dimer (CPD), 8-oxoguanine (8-oxo-G), O6-methylguanine (O6-Me-G), or a single mismatch. Human whole cell extracts could efficiently excise 6-4PPs and cholesterol in an XPC-HR23B-dependent manner, but not 8-oxo-G, O6-Me-G or mismatches. Thus, there was good correlation between the binding specificity of XPC-HR23B for certain types of lesion and the ability of human cell extracts to excise these lesions, supporting the model that XPC-HR23B initiates global genomic NER. Although, XPC-HR23B does not preferentially bind to CPDs, the excision of CPDs in human whole cell extracts was found to be absolutely dependent on XPC-HR23B, in agreement with the in vivo observation that CPDs are not removed from the global genome in XP-C mutant cells. These results suggest that, in addition to the excision repair pathway initiated by XPC-HR23B, there exists another sub-pathway for the global genomic NER that still requires XPC-HR23B but is not initiated by XPC-HR23B. Possible mechanisms will be discussed.
Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Enzimas Reparadoras do DNA , Humanos , Modelos Genéticos , Ligação ProteicaRESUMO
We previously constructed the cell-free nucleotide excision repair (NER) assay system with UV-irradiated SV40 minichromosomes to analyze the mechanism of NER reaction on chromatin DNA. Here we investigate the factor that acts especially on nucleosomal DNA during the damage excision reaction, and reconstitute the damage excision reaction on SV40 minichromosomes. NER-proficient HeLa whole cell extracts were fractionated, and the amounts of known NER factors involved in the column fractions were determined by immunoblot analyses. The column fractions were quantitatively and systematically replaced by highly purified NER factors. Finally, damage DNA excision reaction on SV40 minichromosomes was reconstituted with six highly purified NER factors, XPA, XPC-HR23B, XPF-ERCC1, XPG, RPA and TFIIH, as those essential for the reaction with naked DNA. Further analysis showed that the damages on chromosomal DNA were excised as the same efficiency as those on naked DNA for short incubation. At longer incubation time, however, the damage excision efficiency on nucleosomal DNA was decreased whereas naked DNA was still vigorously repaired. These observations suggest that although the six purified NER factors have a potential to eliminate the damage DNA from SV40 minichromosomes, the chromatin structure may still have some repressive effects on NER.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição TFII , Fatores de Transcrição/metabolismo , Dano ao DNA , DNA Viral , Endonucleases , Células HeLa , Humanos , Immunoblotting/métodos , Proteínas Nucleares , Proteína de Replicação A , Vírus 40 dos Símios/genética , Fator de Transcrição TFIIH , Proteína de Xeroderma Pigmentoso Grupo ARESUMO
Simian virus 40 minichromosomes were irradiated with ultraviolet light and used to study cell-free DNA repair reactions by soluble extracts of repair-proficient human cells. Damage-specific incorporation of labeled nucleotides was measured after linearization and electrophoresis of the viral DNA. Repair synthesis with irradiated chromosomes was dependent on the dose of ultraviolet light, but naked viral DNA irradiated at the same doses showed significantly higher template activity. The reactions absolutely required the presence of cell extract, but an extract from Chinese hamster cells was as active as those from human cells. The repair activity was apparently stimulated by the addition of unirradiated plasmid DNA, which did not affect the nucleosome density of the viral chromosomes. ATP and its regenerating system were required for the reaction, but total repair synthesis was little affected by exogenous addition of substrates for RNA synthesis and poly(ADP-ribosyl)ation. Most of the repaired viral chromosomal DNA was in the supercoiled, closed circular form, suggesting that excision repair reactions proceeded to the stage of ligation in the present cell-free system.
Assuntos
Cromossomos/fisiologia , Reparo do DNA , DNA Viral/genética , Vírus 40 dos Símios/genética , Raios Ultravioleta , Animais , Células CHO , Linhagem Celular , Sistema Livre de Células , Cromossomos/efeitos da radiação , Cricetinae , Dano ao DNA , DNA Viral/efeitos da radiação , Haplorrinos , Humanos , Cinética , Cloreto de Magnésio/farmacologia , Plasmídeos , Cloreto de Potássio/farmacologiaRESUMO
Platinum anticancer agents form bulky DNA adducts which are thought to exert their cytotoxic effect by blocking DNA replication. Translesion synthesis, one of the pathways of postreplication repair, is thought to account for some resistance to DNA damage and much of the mutagenicity of bulky DNA adducts in dividing cells. Oxaliplatin has been shown to be effective in cisplatin-resistant cell lines and less mutagenic than cisplatin in the Ames assay. We have shown that the eukaryotic DNA polymerases yeast pol zeta, human pol beta, and human pol gamma bypass oxaliplatin-GG adducts more efficiently than cisplatin-GG adducts. Human pol eta, a product of the XPV gene, has been shown to catalyze efficient translesion synthesis past cis, syn-cyclobutane pyrimidine dimers. In the present study we compared translesion synthesis past different Pt-GG adducts by human pol eta. Our data show that, similar to other eukaryotic DNA polymerases, pol eta bypasses oxaliplatin-GG adducts more efficiently than cisplatin-GG adducts. However, pol eta-catalyzed translesion replication past Pt-DNA adducts was more efficient and less accurate than that seen for previously tested polymerases. We show that the efficiency and fidelity of translesion replication past Pt-DNA adducts appear to be determined by both the structure of the adduct and the DNA polymerase active site.
Assuntos
Cisplatino/metabolismo , Ilhas de CpG/genética , Adutos de DNA/metabolismo , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Compostos Organoplatínicos/metabolismo , Sequência de Bases , DNA/biossíntese , DNA/genética , DNA/metabolismo , Adutos de DNA/genética , Primers do DNA/genética , Primers do DNA/metabolismo , Humanos , Cinética , Ligantes , Oxaliplatina , Dímeros de Pirimidina/genética , Dímeros de Pirimidina/metabolismo , Especificidade por Substrato , Moldes Genéticos , Termodinâmica , DNA Polimerase iotaRESUMO
The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta (pol eta), which is involved in the replication of damaged DNA. Pol eta catalyzes efficient and accurate translesion synthesis past cis-syn cyclobutane di-thymine lesions. Here we show that human pol eta can catalyze translesion synthesis past an abasic (AP) site analog, N-2-acetylaminofluorene (AAF)-modified guanine, and a cisplatin-induced intrastrand cross-link between two guanines. Pol eta preferentially incorporated dAMP and dGMP opposite AP, and dCMP opposite AAF-G and cisplatin-GG, but other nucleotides were also incorporated opposite these lesions. However, after incorporating an incorrect nucleotide opposite a lesion, pol eta could not continue chain elongation. In contrast, after incorporating the correct nucleotide opposite a lesion, pol eta could continue chain elongation, whereas pol alpha could not. Thus, the fidelity of translesion synthesis by human pol eta relies not only on the ability of this enzyme to incorporate the correct nucleotide opposite a lesion, but also on its ability to elongate only DNA chains that have a correctly incorporated nucleotide opposite a lesion.
Assuntos
Dano ao DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Xeroderma Pigmentoso/genética , DNA Polimerase I/metabolismo , Desoxirribonucleotídeos/metabolismo , Guanina/análogos & derivados , Humanos , Mutagênese , DNA Polimerase iotaRESUMO
HeLa cell proteins that bind DNA containing the pyrimidine(6-4)pyrimidone photoproduct were detected by the electrophoretic mobility shift assay using synthetic oligonucleotide duplexes as probes. The major species was purified to near homogeneity, and the amino acid sequences of the proteolytic peptides revealed that it was the human damage-specific DNA-binding protein, which was reported previously. The substrate specificity of this protein was determined using damaged or modified DNA duplexes.
Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/metabolismo , Dímeros de Pirimidina , Sequência de Bases , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/isolamento & purificação , Células HeLa , Humanos , Dados de Sequência MolecularRESUMO
We have constructed a cell-free DNA repair system with UV-irradiated SV40 minichromosomes, as described in the accompanying paper (Sugasawa, K, Masutani, C., and Hanaoka, F. (1993) J. Biol. Chem 268, 9098-9104). In this study, we examined DNA repair synthesis by cell extracts from seven xeroderma pigmentosum (XP) complementation groups, A through G. DNA repair synthesis by XP cell extracts was lower than that with repair-proficient human 293 cell extract and did not increase to the level with the latter on increase in the amount of cell extract or the incubation time. The defects of XP cell extracts were complemented by addition of extracts from cells of different complementation groups, indicating that defective proteins in XP-A through G cells are directly involved in DNA repair. Addition of T4 endonuclease V, which is reported to complement defects of XP cells, stimulated DNA repair synthesis by the 293 cell extract, and also complemented the defects of all XP cell extracts. The XPAC gene product was shown to be involved in DNA repair synthesis using anti-xpac serum and xpac protein produced in Escherichia coli. Anti-xpac serum inhibited DNA repair synthesis by the 293 cell extract and xpac protein reversed the inhibition. Furthermore, xpac protein complemented the defects of extracts of two lines of XP-A cells (XP2OSSV and XP12ROSV) but had no effect on the reactions of extracts from cells of other complementation groups. These findings are consistent with previous results obtained in experiments with cells, indicating that our system is useful for analyzing the mechanisms of DNA excision repair in mammalian cells.
Assuntos
Reparo do DNA , DNA Viral/genética , Vírus 40 dos Símios/genética , Raios Ultravioleta , Xeroderma Pigmentoso/genética , Linhagem Celular , Linhagem Celular Transformada , Sistema Livre de Células , Cromossomos/efeitos da radiação , Dano ao DNA , DNA Viral/biossíntese , DNA Viral/efeitos da radiação , Teste de Complementação Genética , HumanosRESUMO
The mouse DNA primase-DNA polymerase alpha complex can be resolved with buffer containing 50% ethylene glycol (Suzuki, M., Enomoto, T., Hanaoka, F., and Yamada, M. (1985) J. Biochem. (Tokyo) 98, 581-584). The dissociated primase and DNA polymerase alpha have been purified sufficiently that there was no cross-contamination with each other. By the use of thus isolated DNA primase and DNA polymerase alpha in addition to DNA primase-DNA polymerase alpha complex, we have studied primer RNA synthesis and DNA elongation separately as well as the coupled reaction of the initiation and elongation of DNA chains. In the absence of deoxyribonucleoside triphosphates, the isolated primase synthesized oligoribonucleotides of an apparent length of 7-11 nucleotides (monomeric oligomer) and multiples of a modal length of 9-10 nucleotides (multimeric oligomer) and fd phage single-stranded circular DNA. Monomeric and dimeric oligomers were synthesized processively, and trimeric and larger oligomers were produced by repeated cycles of processive synthesis. The primase complexed with DNA polymerase alpha mainly synthesized monomeric and a small amount of dimeric oligomers. In the presence of deoxyribonucleoside triphosphates at concentrations above 10 microM, the DNA primase-DNA polymerase alpha complex exclusively synthesized monomeric oligomers only, which were utilized as primers for DNA synthesis. On the other hand, the products synthesized by the isolated primase were qualitatively unchanged as compared with those synthesized in the absence of DNA precursors. When the synthesis of oligomers by the isolated primase was coupled with DNA elongation by the addition of the primase-free DNA polymerase alpha, the synthesis of dimeric oligomers was inhibited as a result of efficient DNA elongation from monomeric oligomers.
Assuntos
Replicação do DNA , RNA Nucleotidiltransferases/isolamento & purificação , Animais , Linhagem Celular , Centrifugação com Gradiente de Concentração , Cromatografia DEAE-Celulose , DNA Primase , Camundongos , Oligodesoxirribonucleotídeos/biossíntese , RNA Nucleotidiltransferases/biossíntese , RNA Nucleotidiltransferases/metabolismo , Moldes GenéticosRESUMO
Two forms of DNA primase stimulatory factor have been purified from mouse FM3A cells and shown to have RNase H activity. One of the factors, which consists of three polypeptides of 42,000, 41,000, and 27,000 daltons, was characterized in its properties as RNase H and DNA primase stimulatory factor. The nucleolytic activity of the factor specifically digested the RNA component of RNA-DNA hybrids in an endonucleolytic manner. The stimulation by the factor was observed in DNA synthesis by DNA primase-DNA polymerase alpha complex on unprimed DNA templates, and the DNA chains synthesized under these conditions in the presence of the factor were much shorter than those synthesized in its absence. The stimulatory effect of the factor on DNA primase activity was directly confirmed with DNA primase dissociated from DNA polymerase alpha by the observation of the increase in the number of synthesized oligoribonucleotides. The primer RNA synthesis by DNA primase-DNA polymerase alpha complex under the condition where DNA synthesis occurred was also significantly stimulated by the factor. Furthermore, under these conditions RNA primers were removed from DNA chains by the RNase H activity of the factor.