Coordinated protein and DNA remodeling by human HLTF on stalled replication fork

Abstract

Human helicase-like transcription factor (HLTF) exhibits ubiquitin ligase activity for proliferating cell nuclear antigen (PCNA) polyubiquitylation as well as double-stranded DNA translocase activity for remodeling stalled replication fork by fork reversal, which can support damage bypass by template switching. However, a stalled replication fork is surrounded by various DNA-binding proteins which can inhibit the access of damage bypass players, and it is unknown how these proteins become displaced. Here we reveal that HLTF has an ATP hydrolysis-dependent protein remodeling activity, by which it can remove proteins bound to the replication fork. Moreover, we demonstrate that HLTF can displace a broad spectrum of proteins such as replication protein A (RPA), PCNA, and replication factor C (RFC), thereby providing the first example for a protein clearing activity at the stalled replication fork. Our findings clarify how remodeling of a stalled replication fork can occur if it is engaged in interactions with masses of proteins.

Fig. 1.

Fork reversal activity of HLTF on modeled replication fork bound by dsDNA-binding protein. (A) Schematic representation of a possible mechanism through which HLTF can coordinately remodel a model replication fork bound by E111Q EcoRI. (B) Gel retardation assay showing sequence-specific binding and formation of stable DNA-protein complex by E111Q EcoRI on oligo-based fork-like structures. Increasing amount of E111Q EcoRI was incubated with homologous fork containing an EcoRI binding site. E111Q EcoRI binding to both the arms of the fork is shown in I, whereas II and III show binding to lagging or leading arm only. (C) Comparison of HLTF and BLM fork reversal activities on homologous fork bound by E111Q EcoRI protein on both the arms. In I, control without BLM or HLTF; II, activity of HLTF on naked fork; III, activity of HLTF on E111Q EcoRI-bound fork; IV, activity of BLM on naked fork; V, activity of BLM on E111Q EcoRI-bound fork. Each lane within the panel represents time points at which samples are collected and are noted at the bottom of the gel. Quantitation is shown in Fig. S1.

Fig. 2.

Evidence for dsDNA-binding protein disposal from DNA by HLTF. (A) Experimental setup to prove the actual displacement of dsDNA-binding protein during fork reversal. A homologous fork with a single EcoRI-binding site is bound to NeutrAvidin beads through its biotin tag, and the E111Q EcoRI displaced from the fork is trapped by a 75-mer labeled duplex containing a single EcoRI site. The trap DNA is subjected to gel retardation assay to confirm the binding of E111Q EcoRI. (B) Gel retardation assay showing trapped E111Q EcoRI displaced from a modeled fork. Lanes 1–2 no protein control, 3–4 HLTF ATPase mutant, 5–6 HLTF wild-type protein. Samples were collected at 0 and 20 min and incubated with duplex trap DNA prior to gel retardation assay. (C) Similar assay like in B, except that instead of a modeled fork a 75/30-mer partial duplex DNA was used. Lanes 1–3 no protein control, 4–6 HLTF ATPase mutant, 7–9 HLTF wild-type protein. Samples were collected at 0, 10, and 20 min for each protein sample and incubated with duplex trap DNA prior to gel retardation assay.

Fig. 3.

Fork reversal activity of HLTF on gapped fork bound by replicative proteins. (A) Fork reversal activity of HLTF on RPA- or SSB-bound substrate. In I, control without HLTF; II, gapped fork without any ssDNA-binding protein; III, RPA bound to gapped fork; IV, SSB bound to gapped fork. Gel shift experiment for confirming RPA and SSB binding to fork DNA is shown in Fig. S4 A and B, respectively. (B) Gel retardation assays for confirming the binding of RPA, PCNA, and RFC to a homologous fork containing a 15-nt gap on its leading arm. Various combinations of RPA (160 nM), PCNA (80 nM), and RFC (80 nM) were incubated with a 15-nt gapped fork. (C) Comparison of HLTF and BLM fork reversal activities on PCNA-, RFC-, and RPA-bound replication-like structures. In I, control without BLM or HLTF; II, activity of HLTF on naked fork; III, activity of HLTF on PCNA-, RFC-, and RPA-bound fork; IV, activity of BLM on naked fork; V, activity of BLM on PCNA-, RFC-, and RPA-bound fork.

Fig. 4.

Model for the role of HLTF in remodeling protein-covered stalled replication forks. (A) Stalled replication fork at an unrepaired DNA lesion has to undergo severe remodeling such that it can clear up most of its protein content, allowing other DNA repair machinery to get access to the DNA. We suggest that one such mechanism through which a fork can clear up its protein content can be facilitated by HLTF. HLTF can have dual functions because it can not only clear up the proteins, but also can give rise to a four-way junction intermediate called chicken foot. This four-way junction can then be used by other subsequent repair pathways like (i) Holliday junction resolvases, which can resolve a four-way junction through their nuclease activity, (ii) template switch-dependent DNA synthesis, where a DNA polymerase extends the 3′ OH end of the leading nascent strand by copying from the nascent lagging strand, (iii) nucleotide excision repair pathway, where a short ssDNA segment is removed creating a single-strand gap in the DNA, which is subsequently filled in by DNA polymerase using the undamaged strand as template. Once the lesion is repaired or bypassed, the stalled replication fork readopts its original structure and the progression of DNA replication is reestablished.