DNA polymerase delta and epsilon holoenzymes from calf thymus.
Replication of singly-DNA primed M13 DNA by DNA polymerase (pol) delta completely relies on the simultaneous addition of proliferating cell nuclear antigen (PCNA), replication factor C (RF-C) and replication protein A (RP-A) (or E. coli single-strand DNA binding protein, SSB). Pol epsilon core alone is able to synthesize the products on singly-primed ssDNA. However, DNA synthesis by pol epsilon was stimulated up to 10-fold upon addition of the three auxiliary proteins PCNA, RF-C and SSB. This stimulation of pol epsilon by PCNA/RF-C/SSB appears to be the superposition of two events: pol epsilon holoenzyme (pol epsilon, PCNA, RF-C) synthesized longer products than its pol epsilon core counterpart, but elongated less primers. Furthermore, we analyzed the cooperative action of pol alpha/primase with pol delta or pol epsilon holoenzymes on unprimed M13 DNA. While pol delta displayed higher dNMP incorporation than pol epsilon, when a single primer was preannealed to DNA, pol epsilon was more efficient in the utilization of the primers synthesized by pol alpha/primase. Under these conditions both longer products and a higher amount of dNMP incorporation was found for pol epsilon holoenzyme, than for pol delta. Our data support the hypothesis of pol delta as the leading and pol epsilon as the second lagging strand replication enzyme.
Department of Pharmacology and Biochemistry, University Zürich-Irchel, Switzerland.
V
V
Podust
V
V
Mikhailov
A
A
Georgaki
U
U
Hübscher
D000818:Animals; D002417:Cattle; D004257:DNA Polymerase II / Q000502:physiology; D004258:DNA Polymerase III; D019915:DNA Primase; D004261:DNA Replication / Q000502:physiology*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000502:physiology; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D018398:Homeodomain Proteins; D015778:Minor Histocompatibility Antigens; D009687:Nuclear Proteins / Q000502:physiology; D018809:Proliferating Cell Nuclear Antigen; D019253:Proto-Oncogene Proteins c-bcl-2; D012316:RNA Nucleotidyltransferases / Q000502:physiology; D051818:Replication Protein C; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D013950:Thymus Gland / Q000201:enzymology
C096816:BCL2-related protein A1; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C096063:MATA1 protein, S cerevisiae; D015778:Minor Histocompatibility Antigens; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D019253:Proto-Oncogene Proteins c-bcl-2; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D051818:Replication Protein C