Localization of the Deoxyribose Phosphate Lyase Active Site in Human DNA Polymerase ι by Controlled Proteolysis

Human DNA polymerase ι (pol ι) 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 ι has deoxyrib...

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Published in:The Journal of biological chemistry 2003-08, Vol.278 (32), p.29649-29654
Main Authors: Prasad, Rajendra, Bebenek, Katarzyna, Hou, Esther, Shock, David D., Beard, William A., Woodgate, Roger, Kunkel, Thomas A., Wilson, Samuel H.
Format: Article
Language:English
Subjects:
Binding Sites
Chymotrypsin - pharmacology
Cross-Linking Reagents - pharmacology
DNA Polymerase beta - metabolism
DNA Repair
DNA-Directed DNA Polymerase - chemistry
DNA-Directed DNA Polymerase - metabolism
Glutathione Transferase - metabolism
Humans
Kinetics
Phosphorus-Oxygen Lyases - chemistry
Phosphorus-Oxygen Lyases - metabolism
Protein Binding
Protein Structure, Tertiary
Subcellular Fractions
Time Factors
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Summary:Human DNA polymerase ι (pol ι) 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 ι 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 ι 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 ∼Met445. A covalently cross-linked pol ι-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 ι was able to complement the in vitro single-nucleotide BER deficiency of a DNA polymerase β null cell extract.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M305399200