title: DNA polymerase λ inactivation by oxidized abasic sites.
abstract: Base excision repair (BER) plays a vital role in maintaining genomic integrity in mammalian cells. DNA polymerase λ (Pol λ) is believed to play a backup role to DNA polymerase β (Pol β) in base excision repair. Two oxidized abasic lesions that are produced by a variety of DNA-damaging agents, including several antitumor antibiotics, the C4'-oxidized abasic site following Ape1 incision (pC4-AP), and 5'-(2-phosphoryl-1,4-dioxobutane) (DOB), irreversibly inactivate Pol β and Pol λ. The interactions of DOB and pC4-AP with Pol λ are examined in detail using DNA substrates containing these lesions at defined sites. Single-turnover kinetic experiments show that Pol λ excises DOB almost 13 times more slowly than a 5'-phosphorylated 2-deoxyribose (dRP). pC4-AP is excised approximately twice as fast as DOB. The absolute rate constants are considerably slower than those reported for Pol β for the respective reactions, suggesting that Pol λ may be an inefficient backup in BER. DOB inactivates Pol λ approximately 3-fold less efficiently than it does Pol β, and the difference can be attributed to a higher K(I) (33 ± 7 nM). Inactivation of Pol λ's lyase activity by DOB also prevents the enzyme from conducting polymerization following preincubation of the protein and DNA. Mass spectral analysis of GluC-digested Pol λ inactivated by DOB shows that Lys324 is modified. There is inferential support for the idea that Lys312 may also be modified. Both residues are within the Pol λ lyase active site. When acting on pC4-AP, Pol λ achieves approximately four turnovers on average before being inactivated. Lyase inactivation by pC4-AP is also accompanied by loss of polymerase activity, and mass spectrometry indicates that Lys312 and Lys324 are modified by the lesion. The ability of DOB and pC4-AP to inactivate Pol λ provides additional evidence that these lesions are significant sources of the cytotoxicity of DNA-damaging agents that produce them.
authors: Department of Chemistry, Johns Hopkins University , 3400 North Charles Street, Baltimore, MD 21218, United States.
authors: Adam J
authors: AJ
authors: Stevens
authors: Lirui
authors: L
authors: Guan
authors: Katarzyna
authors: K
authors: Bebenek
authors: Thomas A
authors: TA
authors: Kunkel
authors: Marc M
authors: MM
authors: Greenberg
mesh_terms: D001483:Base Sequence; D002074:Butanones / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D019951:DNA Polymerase beta / Q000378:metabolism*; D003855:Deoxyribose / Q000031:analogs & derivatives* / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D010084:Oxidation-Reduction
chemical_list: C546811:5'-(2-phosphoryl-1,4-dioxobutane); D002074:Butanones; D003855:Deoxyribose; D004247:DNA; C415367:DNA polymerase beta2; D019951:DNA Polymerase beta