|
Repair proteins alter the local DNA structure during nucleotide excision repair
(NER). However, the precise role of DNA melting remains unknown. A series of DNA
substrates containing a unique site-specific BPDE-guanine adduct in a region of
non-complementary bases were examined for incision by the Escherichia coli UvrBC
endonuclease in the presence or absence of UvrA. UvrBC formed a pre-incision
intermediate with a DNA substrate containing a 6-base bubble structure with 2
unpaired bases 5' and 3 unpaired bases 3' to the adduct. Formation of this
bubble served as a dynamic recognition step in damage processing. UvrB or UvrBC
may form one of three stable repair intermediates with DNA substrates, depending
upon the state of the DNA surrounding the modified base. The dual incisions were
strongly determined by the distance between the adduct and the
double-stranded-single-stranded DNA junction of the bubble, and required
homologous double-stranded DNA at both incision sites. Remarkably, in the
absence of UvrA, UvrBC nuclease can make both 3' and 5' incisions on substrates
with bubbles of 3-6 nucleotides, and an uncoupled 5' incision on bubbles of
>/=>/=10 nucleotides. These data support the hypothesis that the E.coli and
human NER systems recognize and process DNA damage in a highly conserved manner.
|
