DNA polymerase beta-dependent long patch base excision repair in living cells.
We examined a role for DNA polymerase beta (Pol beta) in mammalian long patch base excision repair (LP BER). Although a role for Pol beta is well known in single-nucleotide BER, information on this enzyme in the context of LP BER has been limited. To examine the question of Pol beta involvement in LP BER, we made use of nucleotide excision repair-deficient human XPA cells expressing UVDE (XPA-UVDE), which introduces a nick directly 5' to the cyclobutane pyrimidine dimer or 6-4 photoproduct, leaving ends with 3'-OH and 5'-phosphorylated UV lesion. We observed recruitment of GFP-fused Pol beta to focal sites of nuclear UV irradiation, consistent with a role of Pol beta in repair of UV-induced photoproducts adjacent to a strand break. This was the first evidence of Pol beta recruitment in LP BER in vivo. In cell extract, a 5'-blocked oligodeoxynucleotide substrate containing a nicked 5'-cyclobutane pyrimidine dimer was repaired by Pol beta-dependent LP BER. We also demonstrated Pol beta involvement in LP BER by making use of mouse cells that are double null for XPA and Pol beta. These results were extended by experiments with oligodeoxynucleotide substrates and purified human Pol beta.
Asagoshi
Kenjiro
K
Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Liu
Yuan
Y
Masaoka
Aya
A
Lan
Li
L
Prasad
Rajendra
R
Horton
Julie K
JK
Brown
Ashley R
AR
Wang
Xiao-hong
XH
Bdour
Hussam M
HM
Sobol
Robert W
RW
Taylor
John-Stephen
JS
Yasui
Akira
A
Wilson
Samuel H
SH
D000818:Animals; D001483:Base Sequence; D002457:Cell Extracts; D002460:Cell Line; D002470:Cell Survival / Q000528:radiation effects; D019951:DNA Polymerase beta / Q000302:isolation & purification / Q000378:metabolism*; D004260:DNA Repair / Q000528:radiation effects; D004720:Endonucleases / Q000378:metabolism; D045585:Flap Endonucleases / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding / Q000528:radiation effects; D011740:Pyrimidine Dimers / Q000378:metabolism; D014466:Ultraviolet Rays; D051760:Xeroderma Pigmentosum Group A Protein / Q000378:metabolism
D002457:Cell Extracts; D009841:Oligonucleotides; D011740:Pyrimidine Dimers; D051760:Xeroderma Pigmentosum Group A Protein; D019951:DNA Polymerase beta; D004720:Endonucleases; D045585:Flap Endonucleases; C445669:UVDE protein, Neurospora crassa