Nature | Vol 585 | 24 September 2020 | 609Article
Bridging of DNA breaks activates PARP2–HPF1
to modify chromatin
Silvija Bilokapic^1 , Marcin J. Suskiewicz^2 , Ivan Ahel^2 & Mario Halic^1 ✉Breaks in DNA strands recruit the protein PARP1 and its paralogue PARP2 to modify
histones and other substrates through the addition of mono- and poly(ADP-ribose)
(PAR)^1 –^5. In the DNA damage responses, this post-translational modification occurs
predominantly on serine residues^6 –^8 and requires HPF1, an accessory factor that
switches the amino acid specificity of PARP1 and PARP2 from aspartate or glutamate
to serine^9 ,^10. Poly(ADP) ribosylation (PARylation) is important for subsequent chromatin
decompaction and provides an anchor for the recruitment of downstream signalling
and repair factors to the sites of DNA breaks^2 ,^11. Here, to understand the molecular
mechanism by which PARP enzymes recognize DNA breaks within chromatin, we
determined the cryo-electron-microscopic structure of human PARP2–HPF1 bound to
a nucleosome. This showed that PARP2–HPF1 bridges two nucleosomes, with the broken
DNA aligned in a position suitable for ligation, revealing the initial step in the repair of
double-strand DNA breaks. The bridging induces structural changes in PARP2 that
signal the recognition of a DNA break to the catalytic domain, which licenses HPF1
binding and PARP2 activation. Our data suggest that active PARP2 cycles through
different conformational states to exchange NAD+ and substrate, which may enable
PARP enzymes to act processively while bound to chromatin. The processes of PARP
activation and the PARP catalytic cycle we describe can explain mechanisms of
resistance to PARP inhibitors and will aid the development of better inhibitors as
cancer treatments^12 –^16.To visualize the recognition of double-strand DNA lesions, we assem-
bled the complex of human PARP2–HPF1 bound to a mononucleo-
some, which mimics a double-strand DNA break in a chromatin context
(Extended Data Fig. 1a–c). In the electron micrographs, 2D class aver-
ages and initial 3D reconstructions, we primarily observed two nucle-
osomes held together by an additional density bound at the DNA end of
each nucleosome (Extended Data Figs. 1d, e, 2a, Extended Data Table 1).
To improve the resolution of this flexible assembly, we used focused
classification followed by local search refinements. This improved the
resolution to 2.2 Å for nucleosome 2 (Extended Data Fig. 2b–d), 2.8 Å
for nucleosome 1 (Extended Data Fig. 2e), 4.0 Å for the density bridging
two nucleosomes (Extended Data Fig. 2f, g) and 3.9 Å for PARP2–HPF1
(Extended Data Fig. 3a–h). All maps had sufficient overlapping densi-
ties to allow the assembly of a composite map and model (Fig. 1a, b).
In the structure, PARP2–HPF1 is located between two fully wrapped
nucleosomes^17 and holds them together at the double-strand DNA break
(Fig. 1a, b). This end-to-end DNA bridge is the only contact between the
two nucleosomes. Two PARP2–HPF1 complexes bind at the DNA break
through their WGR domains (Fig. 1a, b); however, because of the flex-
ibility of the entire complex, we focused on improving the resolution
of one PARP2–HPF1. In a subset of data, nonetheless, both copies of
the PARP2–HPF1 complex were present in a defined position (Fig. 1c,
Extended Data Fig. 4a, b).
In the structure, the PARP2–HPF1 catalytic site—which is formed
at the heterodimer interface^18 —is positioned near the H3 N-terminal
tails, ready to modify serine residues within them. Each PARP2–HPF1
is positioned to modify one H3 tail of each of the two neighbouring
nucleosomes (Fig. 1b, c).PARP2–HPF1 bridges two nucleosomes
The WGR domains of PARP2 bind a DNA break and hold two nucle-
osomes together (Fig. 1d, Extended Data Fig. 4c–f ). In our data, all such
bridges were symmetrically bound by the WGR domains of two PARP2
proteins; however, a single WGR domain could bridge the DNA break by
simultaneously binding the juxtaposed 5′-phosphate and 3′ end of the
broken DNA strand. In this assembly, two double-strand DNA breaks are
brought together in a way that facilitates subsequent ligation. We did
not observe direct interaction of two PARP2 enzymes, and two PARP2
WGR domains bound the break independently of each other. Binding
of PARP2 WGR domains to nucleosomal DNA ends is consistent with
an earlier X-ray structure of an isolated PARP2 WGR domain bound to
short DNA fragments^19 (Extended Data Fig. 4e, f ).
Besides interacting with the WGR domains, the PARP2 catalytic
domain and HPF1 make several contacts with the nucleosomal and
linker DNA. The helical domain (HD) loop connecting helices αD andhttps://doi.org/10.1038/s41586-020-2725-7
Received: 16 January 2020
Accepted: 22 June 2020
Published online: 16 September 2020
Check for updates(^1) Department of Structural Biology, St Jude Children’s Research Hospital, Memphis, TN, USA. (^2) Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.
✉e-mail: [email protected]