Preferential interaction of loach DNA polymerase delta with DNA duplexes containing single-stranded gaps.
We studied the interaction of DNA polymerase delta (pol delta) purified from the eggs of the teleost fish Misgurnus fossilis (loach) with DNA duplexes containing single-stranded gaps of 1-13 nucleotides (nt). In the absence of processivity factors (PCNA, RF-C, and ATP), pol delta elongated primers on single-stranded DNA templates in a distributive manner. However, the enzyme was capable of processive synthesis by filling gaps of 5-9 nt in DNA duplexes. These data suggest that, upon filling a small gap, pol delta interacts with the 5'-terminus downstream of the gap as well as with the 3'-terminus of the primer. Interaction of pol delta with the proximal 5'-terminus restricting the gap was confirmed by electrophoretic mobility shift assay. Analysis of the enzyme binding to DNA duplexes containing gaps of various sizes showed a much higher affinity of pol delta for duplexes with gaps of about 10 nt than for DNA substrates with primers annealed to single-stranded templates. The most efficient pol delta binding was observed in experiments with DNA substrates containing unpaired 3'-tails in primers. The data obtained suggest that DNA molecules with small gaps and single-stranded tails may serve as substrates for direct action of pol delta in the course of DNA repair.
Sharova
N P
NP
Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Vavilova St. 26, 117808, Moscow, Russia.
Abramova
E B
EB
Dmitrieva
S B
SB
Dimitrova
D D
DD
Mikhailov
V S
VS
D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D003531:Cypriniformes / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D004258:DNA Polymerase III / Q000378:metabolism*; D015342:DNA Probes / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology; D018398:Homeodomain Proteins; D006868:Hydrolysis; D015778:Minor Histocompatibility Antigens; D018809:Proliferating Cell Nuclear Antigen / Q000502:physiology; D011485:Protein Binding; D019253:Proto-Oncogene Proteins c-bcl-2; D051818:Replication Protein C; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion / Q000235:genetics*; D013379:Substrate Specificity; D013698:Templates, Genetic
C096816:BCL2-related protein A1; D015342:DNA Probes; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C096063:MATA1 protein, S cerevisiae; D015778:Minor Histocompatibility Antigens; D018809:Proliferating Cell Nuclear Antigen; D019253:Proto-Oncogene Proteins c-bcl-2; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D004258:DNA Polymerase III; D051818:Replication Protein C