Dideoxynucleoside triphosphate-sensitive DNA polymerase from rice is involved in base excision repair and immunologically similar to mammalian DNA pol beta.
A single polypeptide with ddNTP-sensitive DNA polymerase activity was purified to near homogeneity from the shoot tips of rice seedlings and analysis of the preparations by SDS-PAGE followed by silver staining showed a polypeptide of 67 kDa size. The DNA polymerase activity was found to be inhibitory by ddNTP in both in vitro DNA polymerase activity assay and activity gel analysis. Aphidicolin, an inhibitor of other types of DNA polymerases, had no effect on plant enzyme. The 67 kDa rice DNA polymerase was found to be recognized by the polyclonal antibody (purified IgG) made against rat DNA polymerase beta (pol beta) both in solution and also on Western blot. The recognition was found to be very specific as the activity of Klenow enzyme was unaffected by the antibody. The ability of rice nuclear extract to correct G:U mismatch of oligo-duplex was observed when oligo-duplex with 32P-labeled lower strand containing U (at 22nd position) was used as substrate. Differential appearance of bands at 21-mer, 22-mer, and 51-mer position in presence of dCTP was visible only with G:U mismatch oligo-duplex, but not with G:C oligo-duplex. While ddCTP or polyclonal antibody against rat-DNA pol beta inhibits base excision repair (BER), aphidicolin had no effect. These results for the first time clearly demonstrate the ability of rice nuclear extract to run BER and the involvement of ddNTP-sensitive pol beta type DNA polymerase. Immunological similarity of the ddNTP-sensitive DNA polymerase beta of rice and rat and its involvement in BER revealed the conservation of structure and function of ddNTP-sensitive DNA pol beta in plant and animal.
Sarkar
Sailendra Nath
SN
Department of Botany, Bose Institute, 93/1, Acharya Prafulla Chandra Road, Kolkata-700 009, India.
Bakshi
Sankar
S
Mokkapati
Sanath K
SK
Roy
Sujit
S
Sengupta
Dibyendu N
DN
D000375:Aging / Q000378:metabolism; D000818:Animals; D019951:DNA Polymerase beta / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000276:immunology*; D004260:DNA Repair / Q000502:physiology*; D015224:Dideoxynucleosides / Q000737:chemistry*; D015226:Dinucleoside Phosphates / Q000737:chemistry*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry; D008322:Mammals; D012275:Oryza / Q000201:enzymology* / Q000276:immunology*; D051381:Rats; D012639:Seeds / Q000201:enzymology / Q000276:immunology
D015224:Dideoxynucleosides; D015226:Dinucleoside Phosphates; D004791:Enzyme Inhibitors; D019951:DNA Polymerase beta