610 | Nature | Vol 585 | 24 September 2020
Article
αE of the PARP2 catalytic HD subdomain interacts with the linker DNA
of the second nucleosome (Fig. 1d). HPF1 also contacts the linker DNA
of the same nucleosome through positively charged residues in helix
α8, and positively charged residues in several loops make contact with
the nucleosomal DNA near the dyad and the entry–exit site (Fig. 1d, e,
Extended Data Fig. 4g). In agreement with the structure, the complex with
nucleosome is more stable than the complex with DNA, and HPF1 contrib-
utes to this stability (Extended Data Fig. 5a, b). Moreover, mutations of
HPF1 residues that bind nucleosomal DNA destabilize the interaction of
HPF1 with the PARP2–nucleosome complex (Extended Data Fig. 5c, d). In
the class of structure with two PARP2–HPF1 bound to the nucleosome, we
observe an additional density that interacts with the DNA bridge and HPF1,
which might be the N-terminal tail of PARP2^20 (Extended Data Fig. 5e).
We did not find a class of structure in which PARP2 was not bound
by HPF1, confirming that when bound to a DNA break in a chromatin
context, these two proteins form a very stable complex. In the
cryo-electron-microscopic (cryo-EM) structure, PARP2 and HPF1
interacted in a fashion similar to that seen in a recent crystal structure^18 ,
with a slight rotation of HPF1 (Extended Data Fig. 5f, g). Notably, PARP2–
HPF1 does not make any contacts with the histone core.
Bridging of DNA break activates PARP2
In our structure, the PARP2–HPF1 complex bridges two nucleosomes,
implying that DNA bridging is required for PARP2 activation. To test this
hypothesis, we bound PARP2 to DNA, which generated three distinct
complexes: PARP2 bound to one DNA, one PARP2 bridging two DNAs
and two PARP2 bridging two DNAs (Fig. 2a, Extended Data Fig. 6a).
Adding NAD+ to these complexes led to specific auto-PARylation of
PARP2 that was bridging two DNAs (Fig. 2a). The assay also reveals
that bridging of the DNA break by a single PARP2 is sufficient to trigger
PARylation, consistent with the observation that each WGR domain
independently bridges the broken DNA strands (Fig. 1d).
Although PARP2 forms a stable bridge with DNA ends bearing
5′-phosphate groups (Figs. 1 , 2a), this bridging is severely compromised
upon loss of phosphates (Extended Data Fig. 6b). The bridged complex,
however, shows similar PARylation activity when bound to either
5′-phosphate or 5′-hydroxyl DNA ends, indicating that weak PARylation
of 5′-hydroxyl DNA^19 ,^21 is a result of inefficient DNA bridging (Extended
Data Fig. 6b–d). Notably, in the bridged complex, R140 in the WGR
signalling loop makes an essential contact with the second nucleosome
DNA, and its mutation abolishes the formation of the complex and
PARylation of H3 (Figs. 1d, 2b, Extended Data Fig. 6e–g). Our data
show that bridging of the double-strand DNA break is required for
DNA-dependent activation of PARP2 and that our structure represents
the activated state of the PARP2–HPF1 complex.
We compared our cryo-EM structure of PARP2–HPF1 bound to nucle-
osomes with X-ray structures of PARP1 bound to DNA (Protein Data
Bank code (PDB) 4DQY), which represents a state before activation^22 ,^23.
In our structure, the hydrophobic and HD loops adopt different con-
formations from those seen in previous X-ray structures^22 ,^24 (Fig. 2c,
Extended Data Fig. 7a–c, Supplementary Video 1). Interactions between
R140 of the WGR signalling loop and the DNA of the second nucleo-
some push this loop—which would otherwise clash with the bridged
DNA—towards the hydrophobic loop connecting helices αB and αD in
the HD subdomain (Fig. 2c, d, Extended Data Fig. 7c). The hydrophobic
and signalling loops move closer and form a hydrophobic pocket, in
which V141 of the signalling loop interacts with L254 and P253 in the
hydrophobic loop (Fig. 2c, e, Extended Data Fig. 7c, d). Consequently,
K143 in the signalling loop interacts with D299 in the HD loop, pushing
the latter towards the DNA (Fig. 2d, Extended Data Fig. 7d). Although
mutation of V141 increased DNA-independent activation of PARP2, itPARP2–HPF1 (3.9 Å)
Nucleosome 2 (2.2 Å)HPF1_1 PARP2_1PARP2_2HPF1_1 PARP2_1PARP2_2Nucleosome 2 Nucleosome 1cHPF1_1 PARP2_1PARP2_2Nucleosome 2 Nucleosome 1H3 tail H3 tailHPF1_2Active siteH3 tail H3 tailPARP2–HPF1 bound to nucleosome 2 PARP2–HPF1 bound to nucleosome3 ′ OH
nucleosome 2PARP2_2 WGRPARP2_1 WGR5 ′ P nucleosome 2
R140 3 ′ OH nucleosome 1K117K187 Y188K170K166HPF1_1 PARP2 CAT_HDQ146dα 8HD loop αD5 ′ P
nucleosome 1R303L302
K253Y119Active siteH3 tailH3 tail H3 tailH3 tailK179 R174T177SHL1α 5 α 4R250Signalling loopK180 K124K149–K150abeNucleosome 1 (2.8 Å)Fig. 1 | PARP2–HPF1 bridges two mononucleosomes. a, Composite cryo-EM
map of PARP2–HPF1 bound to 5′-phosphorylated nucleosome at a resolution of
2.1–3.9 Å. Grey, nucleosomes; violet and pink, PARP2; magenta, HPF1. b, c, Models
of the cryo-EM structures of one (b) and two PARP2–HPF1 molecules (c)
(denoted 1 and 2 in each case) bound to bridged mononucleosomes. In c, the
two PARP2–HPF1 molecules are positioned to modify H3 on opposite sides of
the nucleosomes. d, View at the bridge, with two PARP2 WGR domains
connecting two nucleosomes containing a double-strand DNA break. The two
DNAs are positioned such that the 5′-phosphate (5′ P) of the nucleosome 1 DNA
is aligned with the 3′-hydroxyl (3′ OH) of the nucleosome 2 DNA, and vice versa.
The PARP2 catalytic domain binds the linker DNA through the loop in the HD
subdomain (CAT_HD), and HPF1 binds the linker DNA through helix α8. e, HPF1
binds nucleosomal DNA near the dyad through several positively charged
residues in the N-terminal domain.