Localization of the deoxyribose phosphate lyase active site in human DNA polymerase iota by controlled proteolysis.
Human DNA polymerase iota (pol iota) is a member of the Y-family of low fidelity lesion bypass DNA polymerases. In addition to a probable role in DNA lesion bypass, this enzyme has recently been shown to be required for somatic hypermutation in human B-cells. We found earlier that human pol iota has deoxyribose phosphate (dRP) lyase activity and unusual specificity for activity during DNA synthesis, suggesting involvement in specialized forms of base excision repair (BER). Here, mapping of the domain structure of human pol iota by controlled proteolysis revealed that the enzyme has a 48-kDa NH2-terminal domain and a protease resistant 40-kDa "core domain" spanning residues Met79 to approximately Met445. A covalently cross-linked pol iota-DNA complex, representing a trapped intermediate in the dRP lyase reaction, was subjected to controlled proteolysis. Cross-linking was mapped to the 40-kDa core domain, indicating that the dRP lyase active site is in this region. To further evaluate the BER capacity of the enzyme, the dRP lyase and DNA polymerase activities were characterized on DNA substrates representing BER intermediates, and we found that pol iota was able to complement the in vitro single-nucleotide BER deficiency of a DNA polymerase beta null cell extract.
Prasad
Rajendra
R
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Bebenek
Katarzyna
K
Hou
Esther
E
Shock
David D
DD
Beard
William A
WA
Woodgate
Roger
R
Kunkel
Thomas A
TA
Wilson
Samuel H
SH
D001665:Binding Sites; D002918:Chymotrypsin / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019951:DNA Polymerase beta / Q000378:metabolism; D004260:DNA Repair; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D019761:Phosphorus-Oxygen Lyases / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013347:Subcellular Fractions; D013997:Time Factors; D000094769:DNA Polymerase iota
D003432:Cross-Linking Reagents; D005982:Glutathione Transferase; C423906:5'-deoxyribose phosphate lyase; D019951:DNA Polymerase beta; D004259:DNA-Directed DNA Polymerase; D002918:Chymotrypsin; D019761:Phosphorus-Oxygen Lyases; D000094769:DNA Polymerase iota; C000722070:POLI protein, human