The DNA-Binding Domain of Human PARP-1 Interacts with DNA Single-Strand Breaks as a Monomer through Its Second Zinc Finger

Abstract

Poly(ADP-ribose)polymerase-1 (PARP-1) is a highly abundant chromatin-associated enzyme present in all higher eukaryotic cell nuclei, where it plays key roles in the maintenance of genomic integrity, chromatin remodeling and transcriptional control. It binds to DNA single- and double-strand breaks through an N-terminal region containing two zinc fingers, F1 and F2, following which its C-terminal catalytic domain becomes activated via an unknown mechanism, causing formation and addition of polyadenosine-ribose (PAR) to acceptor proteins including PARP-1 itself. Here, we report a biophysical and structural characterization of the F1 and F2 fingers of human PARP-1, both as independent fragments and in the context of the 24-kDa DNA-binding domain (F1 + F2). We show that the fingers are structurally independent in the absence of DNA and share a highly similar structural fold and dynamics. The F1 + F2 fragment recognizes DNA single-strand breaks as a monomer and in a single orientation. Using a combination of NMR spectroscopy and other biophysical techniques, we show that recognition is primarily achieved by F2, which binds the DNA in an essentially identical manner whether present in isolation or in the two-finger fragment. F2 interacts much more strongly with nicked or gapped DNA ligands than does F1, and we present a mutational study that suggests origins of this difference. Our data suggest that different DNA lesions are recognized by the DNA-binding domain of PARP-1 in a highly similar conformation, helping to rationalize how the full-length protein participates in multiple steps of DNA single-strand breakage and base excision repair.

The zinc-finger domains of PARP1 cooperate to recognize DNA strand breaks

“…We also conducted an EMSA assay with a double-hairpin DNA molecule that provides a single 'nick' as a potential binding site for PARP1. In a previous study 21 it was suggested that this DNA bound only a single PARP1 molecule under the conditions used. While we did observe some binding of PARP1-DBD at a protein:DNA ratio of 1:1, in our hands a protein:DNA ratio of 2:1 was required to fully shift the DNA (FIGURE 5c).…”

“…Two recent studies 20,21 , while observing DNA binding modes consistent with dimerization or oligomerization in some circumstances, were able to define buffer conditions and DNA molecules in which 1:1 protein:DNA complexes predominated. These apparently contradictory observations may be rationalized in terms of PARP1 having (at least) two modes of interaction with DNA -a monomeric low activity interaction providing chromatin localization (FIGURE 6a), and a dimeric (or oligomeric) interaction coupled to strand breakrecognition, that brings PARP1 molecules into close proximity and thereby facilitates activation and trans-modification (FIGURE 6b,c).…”

“…PARP1 dimerisation at sites of DNA damage has been implicated in multiple previous studies of PARP1 activation 1,7,[26][27][28] , although this has not been universally observed 20,21 .…”

“…However, despite recent work [20][21][22] the biochemical and structural mechanism by which PARP1 recognises and is activated by DNA gaps and breaks remains obscure. A substantial literature suggests that PARP1 activation involves dimerisation mediated by N-terminal and central domains of the protein, with consequent automodification occurring in trans as an intermolecular reaction 1,7,[23][24][25][26] .…”

Superstructure of the centromeric complex of TubZR C plasmid partitioning systems

“…We designed a microarray in which each experimental measurement consisted of a 24-bp double-stranded DNA sequence (Fig. 1B), formed by a hairpin stabilized through a tetraloop (22). This 24-base "window" was scanned through the plasmid in successive measurements; a 4-bp shift per measurement was used to conduct a coarse scan of a large area of pBtoxis, and single base pair shifts were then used for a high-resolution scan of the Bt tubC region ( Fig.…”