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Nucleic Acids Res
2021 May 21;499:5003-5016. doi: 10.1093/nar/gkab269.
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HPF1-dependent PARP activation promotes LIG3-XRCC1-mediated backup pathway of Okazaki fragment ligation.
Kumamoto S
,
Nishiyama A
,
Chiba Y
,
Miyashita R
,
Konishi C
,
Azuma Y
,
Nakanishi M
.
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DNA ligase 1 (LIG1) is known as the major DNA ligase responsible for Okazaki fragment joining. Recent studies have implicated LIG3 complexed with XRCC1 as an alternative player in Okazaki fragment joining in cases where LIG1 is not functional, although the underlying mechanisms are largely unknown. Here, using a cell-free system derived from Xenopus egg extracts, we demonstrated the essential role of PARP1-HPF1 in LIG3-dependent Okazaki fragment joining. We found that Okazaki fragments were eventually ligated even in the absence of LIG1, employing in its place LIG3-XRCC1, which was recruited onto chromatin. Concomitantly, LIG1 deficiency induces ADP-ribosylation of histone H3 in a PARP1-HPF1-dependent manner. The depletion of PARP1 or HPF1 resulted in a failure to recruit LIG3 onto chromatin and a subsequent failure in Okazaki fragment joining in LIG1-depleted extracts. Importantly, Okazaki fragments were not ligated at all when LIG1 and XRCC1 were co-depleted. Our results suggest that a unique form of ADP-ribosylation signaling promotes the recruitment of LIG3 on chromatin and its mediation of Okazaki fragment joining as a backup system for LIG1 perturbation.
Figure 1. Depletion of LIG1 inhibits Okazaki fragment ligation in Xenopus egg extracts. (A) Control or xLIG1 antibodies were used for immunoprecipitation (I. P.) and immunoprecipitated proteins were analyzed by immunoblotting using the indicated antibodies (n = 2). (B) Sequences of the wild-type and the F8A/F9A mutant PIP-box in xLIG1 are shown. (C) Recombinant wild-type xLIG1–3 × Flag or xLIG1-F8A/F9A-3 × Flag was added to the egg extracts. Immunoprecipitation using anti-Flag antibodies was performed and resultant immunoprecipitates were analyzed by immunoblotting using the indicated antibodies (n = 2). (D) Interphase egg extracts were added with sperm chromatin and incubated in the presence of 30 μM aphidicolin or in its absence (DMSO). Chromatin fractions were isolated and analyzed by immunoblotting using the indicated antibodies (n = 2). (E) Immunodepletion efficiency of xLIG1 from Xenopus egg extract (n = 3). (F) Replication products in mock- or xLIG1-depleted extracts were separated by alkaline agarose gel electrophoresis followed by autoradiography (n = 2). (G) xLIG1-depleted extracts were supplemented with wild-type xLIG1–3 × Flag or xLIG1-K721A-3 × Flag and chromatin was isolated. Purified genomic DNA from chromatin was labeled using exonuclease-deficient Klenow fragment and α-32P dCTP, and separated in a denaturing agarose gel (n = 2).
Figure 2. LIG3-XRCC1 ensures Okazaki fragment ligation in the absence of LIG1. (A) Immunoprecipitation (I. P.) of xLIG3 and xXRCC1 from egg extracts. I. P. was performed with anti-xLIG3 or -xXRCC1 antiserum. Pre-immune serum was also used as a control. Immunoprecipitates were analyzed by immunoblotting using the indicated antibodies (n = 2). (B) Mock-, xLIG1-, xXRCC1- or xLIG1/xXRCC1-depleted extracts were used to replicate sperm chromatin. Genomic DNA was purified and analyzed as in Figure 1G (n = 3). (C) Chromatin-bound proteins from (B) were monitored by immunoblotting using the indicated antibodies (n = 3). (D) xLIG1- and xLIG1/xXRCC1-depleted extracts were used to replicate sperm chromatin. xLIG1/xXRCC1-depleted extracts were supplemented with either buffer (+Buffer), wild-type 3× Flag-xLIG3-xXRCC1-myc, or 3× Flag-xLIG3-xXRCC1-R336AK370A-myc lacking the poly-ADP-ribose binding activity of xXRCC1 (n = 3). (E) Replication products from (D) were analyzed as in Figure 1G (n = 2). (F) xLIG1- and xLIG1/xXRCC1-depleted extracts were used to replicate sperm chromatin. xLIG1/xXRCC1-depleted extracts were supplemented with either buffer (+Buffer), wild-type 3× Flag-xLIG3-xXRCC1-myc, 3× Flag-xLIG3 or 3× Flag-xLIG3 lacking the BRCT domain. Replication products were analyzed as in Figure 1G (n = 2).
Figure 3. LIG1 deficiency causes histone H3 ADP-ribosylation. (A) Mock- and xLIG1-depleted extracts were used for replication of sperm chromatin. xLIG1-depleted extracts were supplemented with wild-type xLIG1 or xLIG1-K721A. Chromatin-bound proteins were analyzed by immunoblotting using the indicated antibodies and pan ADP-ribose (Pan-ADPr) detecting reagent (n = 3). The asterisk indicates a non-specific band. (B) Mock-, xLIG1-, xPARG- and xLIG1/xPARG-depleted extracts were used for replication of sperm nuclei. Chromatin-bound proteins were analyzed as in Figure 3A (n = 2). (C) Chromatin fractions from the indicated immunodepleted extracts were solubilized with MNase followed by immunoprecipitation with anti-histone H3 antibodies after treatment with 1% SDS. The resultant immunoprecipitates were analyzed as in Figure 3A (n = 2). (D) Mock- and xLIG1-depleted extracts were used for replication of sperm nuclei in the presence of 30 μM APH where indicated. Chromatin-bound proteins were analyzed by immunoblotting using the indicated antibodies and pan ADP-ribose detecting reagent (n = 2). (E) Mock- and xLIG1-depleted extracts were used for replication of sperm chromatin. xLIG1-depleted extracts were supplemented with either buffer or recombinant wild-type His10-xARH3 or His10-xARH3-D58N, (0.2–1 μM). Chromatin-bound proteins were analyzed by immunoblotting using the indicated antibodies and pan ADP-ribose detecting reagent (n = 2).
Figure 4. PARP1-HPF1 stimulates histone H3 ADP-ribosylation in LIG1-depleted extracts. (A) Mock-, xLIG1- and xLIG1/xPARP1-depleted extracts were analyzed by immunoblotting using the indicated antibodies. (B) The extracts from (A) were used to replicate sperm nuclei. Chromatin-bound proteins were analyzed by immunoblotting (n = 2). (C) Mock-, xLIG1- and xLIG1/xHPF1-depleted extracts were analyzed by immunoblotting using the indicated antibodies. The asterisk indicates a non-specific band. (D) The extracts from (C) were used to replicate sperm nuclei. Chromatin-bound proteins were analyzed (n = 2). (E) xLIG1/xHPF1-depleted extracts were supplemented with buffer, recombinant His10-xHPF1 (WT), or recombinant mutant His10-xHPF1 (Y251A/R252A) defective for PARP1 binding activity. (F) The extracts from (E) were used to replicate sperm nuclei. Chromatin-bound proteins were analyzed (n = 2).
Figure 5. HPF1-dependent PARP activation promotes alternative Okazaki fragment ligation. (A) Mock-, xLIG1-, xPARP1- and xLIG1/xPARP1-depleted extracts were used for replication of sperm nuclei. Chromatin-bound proteins were analyzed by immunoblotting using indicated antibodies (n = 2). (B) Mock-, xLIG1-, xHPF1- and xLIG1/xHPF1-depleted extracts were used for replication of sperm nuclei. Chromatin-bound proteins were analyzed by immunoblotting using the indicated antibodies (n = 2). (C) xLIG1-, xLIG1/xXRCC1-, xLIG1/xPARP1- or xLIG1/xHPF1-depleted extracts were used to replicate sperm chromatin. Genomic DNA was purified and labeled using exonuclease-deficient Klenow fragments and α-32P dCTP. Samples were separated in a denaturing agarose gel, and the incorporation of radioactivity was monitored by autoradiography (n = 3). (D) Replication products in mock-, xLIG1-, xLIG1/xXRCC1-, xPARP1-, xLIG1/xPARP1-, xHPF1- or xLIG1/xHPF1-depleted extracts were separated by alkaline agarose gel electrophoresis followed by autoradiography (n = 4).
Figure 6. HPF1-dependent PARP activation promotes LIG3-XRCC1-mediated backup pathway of Okazaki fragment ligation. In LIG1-depleted egg extracts, the defective Okazaki fragment ligation results in the formation of nicks on the replicating genome. PARP1 binds to DNA at nicks, and ADP-ribosylation of histone H3 and chromatin-binding proteins is induced in PARP1/HPF1-dependent manner. The LIG3/XRCC1 pathway senses the ADP-ribosylation modification and plays an essential role for the Okazaki fragment joining at the late stage of DNA replication.
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