Abstract
Background:
NucS, new bipolar nuclease acting on branched DNA repair substrates, interacts with proliferating cell nuclear antigen (PCNA).
Results:
PCNA and NucS form a stable 1:1 complex and PCNA directs the activity of NucS toward single-stranded/double-stranded DNA junctions in branched DNA substrates.
Conclusion:
PCNA regulates NucS activity, preventing the nonspecific cleavage of NucS on the chromatin.
Significance:
This study will help understand how PCNA regulates its client enzymes.
Pyrococcus abyssi
NucS is the founding member of a new family of structure-specific DNA endonucleases that interact with the replication clamp proliferating cell nuclear antigen (PCNA). Using a combination of small angle x-ray scattering and surface plasmon resonance analyses, we demonstrate the formation of a stable complex in solution, in which one molecule of the
Pab
NucS homodimer binds to the outside surface of the
Pab
PCNA homotrimer. Using fluorescent labels, PCNA is shown to increase the binding affinity of NucS toward single-strand/double-strand junctions on 5′
and
3′ flaps, as well as to modulate the cleavage specificity on the branched DNA structures. Our results indicate that the presence of a single major contact between the
Pab
NucS and
Pab
PCNA proteins, together with the complex-induced DNA bending, facilitate conformational flexibility required for specific cleavage at the single-strand/double-strand DNA junction.