Human XPA i
s an e
ssential component in the multienzyme nucleotide exci
sion repair (NER)pathway. The
solution
structure of the minimal DNA binding domain of XPA (XPA-MBD: M98-F219)wa
s recently determined [Buchko et al. (1998)
Nucleic Acids Res. 26, 2779-2788, Ikegami et al. (1998)
Nat. Struct. Biol. 5, 701-706] and
shown to con
si
st of a compact zinc-binding core and a loop-richC-terminal
subdomain connected by a linker
sequence. Here, the
solution
structure of XPA-MBD wa
sfurther refined u
sing an entirely new cla
ss of re
straint
s ba
sed on p
seudocontact
shift
s mea
sured in cobalt-
sub
stituted XPA-MBD. U
sing thi
s structure, the
surface of XPA-MBD which interact
s with DNA and afragment of the large
st
subunit of replication protein A (RPA70
s/gifchar
s/Delta.gif" BORDER=0 >C327: M1-Y326) wa
s determined u
singchemical
shift mapping. DNA binding in XPA-MBD wa
s highly localized in the loop-rich
subdomain forDNA
with or without a lesion [dihydrothymidine (dhT) or 6-4-thymidine-cytidine (64TC)], or with DNAin
single- or double-stranded form, indicating that the character of the le
sion it
self i
s not the driving forcefor XPA binding DNA. RPA70
s/gifchar
s/Delta.gif" BORDER=0 >C327 wa
s found to contact region
s in
both the zinc-binding and loop-rich
subdomain
s. Some overlap of the DNA and RPA70
s/gifchar
s/Delta.gif" BORDER=0 >C327 binding region
s wa
s ob
served in theloop-rich
subdomain, indicating a po
ssible cooperative DNA-binding mode between XPA and RPA70
s/gifchar
s/Delta.gif" BORDER=0 >C327.To complement the chemical
shift mapping data, the backbone dynamic
s of free XPA-MBD and XPA-MBD bound to DNA oligomer
s containing dhT or 64TC le
sion
s were inve
stigated u
sing
15N NMRrelaxation data. The dynamic analy
se
s for the XPA-MBD complexe
s with DNA revealed localized increa
se
sand decrea
se
s in
S 2 and an increa
se in the global correlation time. Region
s of XPA-MBD with the large
stincrea
se
s in
S 2 overlapped region
s having the large
st chemical
shift
s change
s upon binding DNA, indicatingthat the loop-rich
subdomain become
s more rigid upon binding DNA. Intere
stingly,
S 2 decrea
sed for
some re
sidue
s in the zinc-binding core upon DNA a
ssociation, indicating a po
ssible concerted
structuralrearrangement on binding DNA.