Redefining
the DNA-Binding Domain of Human XPA
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Abstract
Xeroderma
pigmentosum complementation group A (XPA) protein plays
a critical role in the repair of DNA damage via the nucleotide excision
repair (NER) pathway. XPA serves as a scaffold for NER, interacting
with several other NER proteins as well as the DNA substrate. The
critical importance of XPA is underscored by its association with
the most severe clinical phenotypes of the genetic disorder Xeroderma
pigmentosum. Many of these disease-associated mutations map to the
XPA<sub>98–219</sub> DNA-binding domain (DBD) first reported
∼20 years ago. Although multiple solution NMR structures of
XPA<sub>98–219</sub> have been determined, the molecular basis
for the interaction of this domain with DNA is only poorly characterized.
In this report, we demonstrate using a fluorescence anisotropy DNA-binding
assay that the previously reported XPA DBD binds DNA with substantially
weaker affinity than the full-length protein. In-depth analysis of
the XPA sequence suggested that the original DBD construct lacks critical
basic charge and helical elements at its C-terminus. Generation and
analysis of a series of C-terminal extensions beyond residue 219 yielded
a stable, soluble human XPA<sub>98–239</sub> construct that
binds to a Y-shaped ssDNA–dsDNA junction and other substrates
with the same affinity as the full-length protein. Two-dimensional <sup>15</sup>N–<sup>1</sup>H NMR suggested XPA<sub>98–239</sub> contains the same globular core as XPA<sub>98–219</sub> and
likely undergoes a conformational change upon binding DNA. Together,
our results demonstrate that the XPA DBD should be redefined and that
XPA<sub>98–239</sub> is a suitable model to examine the DNA
binding activity of human XPA