Characterization of the DNA-dependent ATPase and a DNA unwinding activity associated with the yeast DNA polymerase .alpha. complex

Biswas, Esther E.; Ewing, Charles M.; Biswas, Subhasis B.

doi:10.1021/bi00063a013

Cited by 16 publications

(30 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Unpublished data). This is consistent with the lower catalytic activity of the DNA-ATPase compared to that observed for helicases 199 1; Biswas et al, 1993) and the absence of the typical helicase "deadbox" sequences in the deduced amino acid sequences of the Ku 80-and 70-kDa subunits (Reeves & Sthoeger, 1989;Yaneva et al, 1989;Mimori et al, 1990). It seems probable, therefore, that the DNA-ATPase activity has a different function(s) than a helicase in DNA metabolism related to protein/DNA and/or protein/protein interactions.…”

Section: Uuuxgxxxx[gas]k[ste]supporting

confidence: 85%

DNA-Dependent ATPase from HeLa Cells Is Related to Human Ku Autoantigen

Cao¹,

Pitt²,

Leszyk³

et al. 1994

Biochemistry

View full text Add to dashboard Cite

A 150-kDa DNA-dependent ATPase composed of 83/68-kDa subunits was previously reported to cofractionate with a 21S complex of enzymes for DNA synthesis from HeLa cells (Vishwanatha, J. K., & Baril, E. F. (1990) Biochemistry 29, 8753-8759). The DNA-dependent ATPase was purified to electrophoretic homogeneity from a HeLa cell homogenate by a modified procedure that involves subcellular fractionation, poly(ethylene-glycol) precipitation of the combined nuclear extract/cytosol, and chromatography on Q-Sepharose and native and denatured DNA/celluloses followed by Mono-S fast protein liquid chromatography. The purified enzyme showed equimolar amounts of 83- and 68-kDa polypeptides following polyacrylamide gel electrophoresis under denaturing conditions. Sequence analysis of peptide fragments derived from the separated 83- and 68-kDa polypeptides showed 90-100% homology with the corresponding 80- and 70-kDa subunits of human Ku protein. Immunoblot analysis of the ATPase during the course of its purification and immunoprecipitation with antibodies to the 80- and 70-kDa subunits of human Ku protein confirmed the relationship of the 83- and 68-kDa polypeptides of the human DNA-dependent ATPase to the subunits of human Ku protein. Both the 83- and 68-kDa polypeptides are phosphorylated by a DNA-dependent protein kinase that cofractionates with the ATPase. The DNA-dependent ATPase activity is up regulated by phosphorylation.

show abstract

Section: Uuuxgxxxx[gas]k[ste]supporting

confidence: 85%

DNA-Dependent ATPase from HeLa Cells Is Related to Human Ku Autoantigen

Cao¹,

Pitt²,

Leszyk³

et al. 1994

Biochemistry

View full text Add to dashboard Cite

show abstract

“…For example, human RP-A and yeast RP-A bind to pyrimidine-rich strands with a 50-fold higher affinity than purine-rich strands (36). A 90-kDa ATPase associated with the yeast DNA polymerase a-primase complex also showed 3-fold greater activity with oligopyrimidines than with oligopurines of the same size (37). The preference for certain sequences, such as that within the collagen IV bidirectional promoter, may suggest that Al p145 has additional functions.…”

Section: Discussionmentioning

confidence: 99%

Cloning of the large subunit of activator 1 (replication factor C) reveals homology with bacterial DNA ligases.

Burbelo

Utani

Pan

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

We have cloned a gene encoding a DNAbinding protein by Southwestern screening of a murine cDNA library with a double-stranded oligonucleotide containing the sequence from the bidirectional promoter of the al and a2 collagen IV genes. The middle portion of this 1131-amino acid protein has a region homologous to bacterial DNA ligases, and the more carboxyl portion contains several domains homologous to p40, p38, p37, and p36.5 subunits of activator 1 (Al, also called replication factor C), a human replication protein complex. Western blotting revealed that antiserum generated against part of the recombinant protein reacted specifically with the 145-kDa component of the purified human Al complex, indicating that it is the murine counterpart of the Al p145. Characterization of the DNA-binding activity of the recombinant fusion protein by gel mobility-shift assay revealed that it had a preference for a run of pyrimidines on one strand. Deletion analysis using recombinant proteins revealed that the DNA ligase-like domain was required for DNA-binding activity. The finding that the region required for the binding of murine Al p145 to DNA has similarity to a domain found in DNA ligases suggests that this region may be utilized by both proteins in recognizing DNA.DNA replication is a highly controlled process regulated by a large number of proteins and enzymes which form multiple protein-DNA and protein-protein complexes (1). The DNA polymerases involved in replication have the ability to rapidly duplicate very long stretches of DNA without dissociation (1). Reconstitution experiments using purified human and yeast proteins have revealed that the ability of DNA polymerases 8 and e to replicate large stretches of DNA is not inherent in the DNA polymerases but rather is due to a number of associated accessory proteins dedicated to maintaining the processive action of these DNA polymerases (2-4).The use of multisubunit proteins to clamp the DNA polymerase onto primed DNA is not restricted to eukaryotes but is a universal process, also occurring in bacteriophage T4 and Escherichia coli (reviewed in refs. 5-7). In each system a multisubunit polymerase accessory protein complex [eukaryotic activator 1 (Al, also called replication factor C, RF-C), T4 gene products 44/62 (g44/62), E. coli ycomplex] functions to recognize the primed template and load a second polymerase accessory protein [eukaryotic proliferating-cell nuclear antigen (PCNA), T4 g45, E. coli /3] on the DNA in the presence of ATP. In E. coli, the y complex (y66'x subunits) places a dimer of the clamp protein (,8 subunit) on the DNA, which then recruits the DNA polymerase III core (8-10). The (3 dimer has been shown to form a ring-shaped clamp around the DNA (11). In T4, ATP is required for the association of the g44/62 (4:1) complex and the g45 protein on primed DNA, and ATP hydrolysis is required to add the g43 DNA poly-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise...

show abstract

“…Oligonucleotide 1, which is a 50-mer complementary to the 50 bp sequence between nucleotides 6268 and 6317 of M13mp19 ssDNA was labeled at its 3′ end using terminal deoxynucleotidyl transferase. The oligo was hybridized to M13mp19 (Figure 1A) as previously described (Biswas et al 1993b). Excess unhybridized labeled oligonucleotide was removed by spin column (Promega, Madison, WI) purification.…”

Section: Methodsmentioning

confidence: 99%

“…Protein concentrations were estimated according to the method of Bradford (l976), using bovine serum albumin as a standard. The ATPase assays were carried out as previously described (Biswas et al 1993b).…”

Section: Methodsmentioning

confidence: 99%

Purification and Characterization of the DNA Polymerase α Associated Exonuclease: The RTH1 Gene Product

Zhu

Biswas

1997

Biochemistry

Self Cite

View full text Add to dashboard Cite

We report here the purification and mechanistic characterization of a 5'-3' exonuclease associated with DNA polymerase alpha from the yeast Saccharomyces cerevisiae. Earlier, we identified a 5' --> 3' exonuclease activity that copurified with yeast DNA polymerase alpha-primase in a multiprotein complex [Biswas, E. E., et al. (1993) Biochemistry, 32, 3020-3027]. Peptide sequence analysis of the purified 47 kDa exonuclease was carried out, and the peptide sequence was found to be identical to the S. cerevisiae gene YKL510 encoded polypeptide, which is also known as yeast RAD2 homolog 1 or RTH1 nuclease. The native exonuclease also had strong flap endonuclease activity similar to that observed with RTH1 nuclease and homologous yeast (RAD2) and mammalian enzymes. During our studies, we have discovered certain unique features of the mechanism of action of the native RTH1 nuclease. Studies presented here indicated that the exonuclease had specific pause sites during its 5'-3' exonuclease nucleotide excision. These pause sites were easily detected with long (approximately 50 bp) oligonucleotide substrates during exonucleolytic excision by the formation of a discontinuous ladder of excision product. We have further analyzed the mechanism of generation of the pause sites, as they could occur through a number of different pathways. Alignment of the pause sites with the nucleotide sequence of the oligonucleotide substrate indicated that the pause sites were dependent on the nucleotide sequence. Our analysis revealed that RTH1 nuclease pauses predominantly at G:C rich sequences. With poly(dA):oligo(dT)50 as substrate, the exonucleolytic products formed a continuous ladder with no evidence of pausing. The G:C rich DNA sequences are thermodynamically more stable than the A:T rich sequences, which may be in part responsible for pausing of the RTH1 5' --> 3' exonuclease at these sites.

show abstract

Characterization of the DNA-dependent ATPase and a DNA unwinding activity associated with the yeast DNA polymerase .alpha. complex

Cited by 16 publications

References 41 publications

DNA-Dependent ATPase from HeLa Cells Is Related to Human Ku Autoantigen

DNA-Dependent ATPase from HeLa Cells Is Related to Human Ku Autoantigen

Cloning of the large subunit of activator 1 (replication factor C) reveals homology with bacterial DNA ligases.

Purification and Characterization of the DNA Polymerase α Associated Exonuclease: The RTH1 Gene Product

Contact Info

Product

Resources

About