Pre-steady-state binding of damaged DNA by XPC-hHR23B reveals a kinetic mechanism for damage discrimination

Kelly S. Trego, John Turchi

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

The XPC-hHR23B complex (XPC-hHR23B) is a heterodimeric protein required for the initial step of DNA damage recognition in the global nucleotide excision repair (NER) pathway. A strong preference of XPC-hHR23B for UV- and cisplatin-damaged DNA has previously been demonstrated using equilibrium binding assays. To better understand the molecular mechanism of damage recognition by XPC-hHR23B, we carried out the pre-steady-state kinetic analysis of the XPC-hHR23B-DNA interactions using a stopped-flow fluorescence assay. XPC-hHR23B displays a faster kon for cisplatin-and UV-damaged duplex DNA than for undamaged DNA, with additional, minor effects on the αoff rates. XPC-hHR23B has a high affinity for undamaged single-stranded DNA compared to duplex DNA, which can be largely attributed to a high rate of association. However, cisplatin damage on single-stranded DNA reduced the overall level of binding by a factor of 7, with nearly equal contributions from changes to the kon and koff rates. Together, these results support a model for initial damage recognition by XPC-hHR23B that is dependent on structural changes in the DNA, and not adduct chemistry.

Original languageEnglish
Pages (from-to)1961-1969
Number of pages9
JournalBiochemistry
Volume45
Issue number6
DOIs
StatePublished - Feb 14 2006

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Kinetics
DNA
Cisplatin
Single-Stranded DNA
Assays
DNA Adducts
DNA Repair
DNA Damage
Fluorescence
Repair
Nucleotides
Association reactions
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Pre-steady-state binding of damaged DNA by XPC-hHR23B reveals a kinetic mechanism for damage discrimination. / Trego, Kelly S.; Turchi, John.

In: Biochemistry, Vol. 45, No. 6, 14.02.2006, p. 1961-1969.

Research output: Contribution to journalArticle

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