Role of double-stranded DNA translocase activity of human HLTF in replication of damaged DNA

Abstract

Unrepaired DNA lesions can block the progression of the replication fork, leading to genomic instability and cancer in higher-order eukaryotes. In Saccharomyces cerevisiae, replication through DNA lesions can be mediated by translesion synthesis DNA polymerases, leading to error-free or error-prone damage bypass, or by Rad5-mediated template switching to the sister chromatid that is inherently error free. While translesion synthesis pathways are highly conserved from yeast to humans, very little is known of a Rad5-like pathway in human cells. Here we show that a human homologue of Rad5, HLTF, can facilitate fork regression and has a role in replication of damaged DNA. We found that HLTF is able to reverse model replication forks, a process which depends on its double-stranded DNA translocase activity. Furthermore, from analysis of isolated dually labeled chromosomal fibers, we demonstrate that in vivo, HLTF promotes the restart of replication forks blocked at DNA lesions. These findings suggest that HLTF can promote error-free replication of damaged DNA and support a role for HLTF in preventing mutagenesis and carcinogenesis, providing thereby for its potential tumor suppressor role.

FIG. 1.

HLTF reverses but does not dissolve oligonucleotide-based model replication-fork substrates. (A) Possible outcomes of enzymatic manipulation of model replication fork substrates. While fork reversal (I) can occur only on homologous forks which have complementary arms, fork dissolution (II) can happen on both homologous and heterologous forks. Whereas coordinated fork reversal requires specific enzymatic activity that leads only to two double-stranded oligonucleotides, fork dissolution by canonical helicase activity results in single-stranded oligonucleotides. (B) Fork regression activity of HLTF tested on heterologous fork (HetF) (I) and homologous fork (HomF) (II) model substrates. The DNA substrates in the gel are indicated by an arrowhead, while the positions of some of the possible products are shown by arrows. The 3′ ends of oligonucleotides are indicated by half arrows, and the positions of 5′ ³²P labels are marked with asterisks. We note that formation of double-stranded products from HomF and not from HetF was used to indicate fork regression activity without dissolution of the fork. In panel III, the ATPase mutant HLTF DE557,558AA (DE) was examined with the HomF substrates. (C) HLTF activity requires ATP hydrolysis. Cofactor dependence of HLTF (15 nM) was tested on the oligonucleotide-based homologous fork model substrate, where one of the template strands and one of the opposite nascent strand were 5′ ³²P labeled. The indicated ribonucleotides and deoxyribonucleotides were used at 5 mM concentrations in the presence of 5 mM MgCl₂, except in lane 5, where 10 mM EDTA was used instead of MgCl₂.

FIG. 2.

HLTF acts concertedly and possesses branch-migrating activity. (A) Kinetics of HLTF activity in the presence or absence of single-stranded DNA binding proteins. Processing of HomF substrate by HLTF (50 nM) was examined at the indicated time points in the absence (I) or presence of E. coli SSB (40 nM) (II) or T4 gp32 (90 nM) (III) proteins. (B) HLTF can migrate a moveable four-way junction. The X0 junction is static, while the core of the X12 junction, which was flanked with 19- to 20-nucleotide-long heterologies at the end of each arm, is movable. We note that product formation most likely requires spontaneous dissolution of terminal heterologies, which can explain the somewhat weaker activity of HLTF on X12 than on homologous fork substrates. Lane 7, marker Y fork containing O1114/O1115; lane 8, marker Y fork containing O1114/O1116; lane 9, boiled X12. Symbols are as described for Fig. 1.

FIG. 3.

HLTF can regress plasmid-sized model forks. (A) Schematic representation of the generation of joint DNA substrate (pG46B′/pG68AXh) (σ-structure) and the outcome of its HLTF-mediated regression named (α-structure). A, H, R, X, S, F, Y, N, and Xh refer to restriction endonuclease sites AvrII, BamHI, EcoRI, BsaXI, SapI, AflIII, BseYI, AlwNI, and XhoI, respectively. The positions of 5′-³²P labels on the “lagging strand” are marked with asterisks. (B) Fork regression by HLTF is extensive and progressive. The transfer of restriction enzyme sites to the regressed arm by HLTF (50 nM) was followed in the presence of 5 mM ATP/Mg (panel II). The positions of the various restriction products generated by digestion of the regressed fork are indicated. The control set with 5 mM AMP-PNP/Mg shows the background level of spontaneous regression (panel I). Progressivity of the reaction is demonstrated by using pG46B′/pG68A SapI[Het]Xh substrate (panel III) in which a 30-bp sequence heterology was introduced at the SapI site (shown by arrowhead), which resulted in blocked regression beyond the heterology. (C) Fork reversal is not affected by single-stranded DNA-binding proteins. Regression through the EcoRI site was monitored at various HLTF concentrations in the presence or absence of E. coli SSB or T4 gp32 proteins.

FIG. 4.

HLTF tracks along double-stranded DNA. (A) HLTF displaces radioactively labeled triplex-forming oligonucleotide (TFO) from partial triple-stranded DNA in an ATP hydrolysis-dependent manner. In the lanes indicated by mut DE, the ATPase mutant HLTF DE557,558AA was substituted for the wild-type HLTF, while in the lanes labeled AMP-PNP, ATP was replaced by AMP-PNP. We note that in the control reaction using isolated (blunted) triple helix, the TFO displacement was severely impaired, indicating that HLTF has to be loaded onto the dsDNA flanking the triplex forming region. (B) Triplex displacement activity of HLTF depends on the integrity of the double-stranded arm. HLTF at increasing concentration was incubated with intact (I), 3′-strand gapped (II), and 5′-strand gapped (III) DNA substrates which are represented schematically. We note that a 6-nucleotide-long gap on either the 3′ or the 5′ strand inhibited HLTF translocation and consequent TFO displacement. (C) Kinetics of triplex displacement by HLTF. The rate of displacement in the presence of 20 nM HLTF was examined at time points between 0 and 30 min (0, 0.5, 1.0, 1.5, 2.0, 4.0, 8.0, 12.0, 16.0, 20.0, 24.0, and 30.0 min) on intact (I), 3′-strand gapped (II), and 5′-strand gapped (III) DNA substrates as represented schematically. Note that some displacement occurred on the substrate with a gap on the 5′ strand but the 3′ gapped substrate was left unprocessed. These properties could classify HLTF as a 3′-5′ dsDNA translocase.

FIG. 5.

HLTF is required for efficient replication fork restart on damaged DNA. (A) Protocol for defining sites and speeds of replication fork before and after treatment by MMS. (B) Schematic representation of possible fiber-labeling patterns. Arrows indicate the direction of fork movements. (C) Images of various types of labeled chromosome fibers. We note that ongoing forks are dually labeled by IdU and BrdU while forks that have stalled upon encountering a DNA lesion during MMS treatment are labeled only by IdU. (D) Effects of HLTF knockdown on MMS-induced stalling of replication forks in siRNA-transfected HeLa cell lines. For HLTF knockdown, HeLa cells were transfected with negative control or HLTF-specific siRNA. For complementation, the HLTF siRNA knocked-down cells were subsequently transfected by siRNA-resistant wild-type, ATPase mutant, RING mutant, or ATPase and RING double-mutant HLTF-expressing plasmids. The percentage of stalled forks was calculated on the basis of the sum of stalled and ongoing forks being 100%. (E) Effect of MMS treatment on replication movement. Apparent average fork rates were calculated by dividing the length of labeled fiber with the labeling time for both halogenated nucleotides. The ratio of apparent average fork rates after and before MMS treatment was followed in a 20- to 90-min period following MMS treatment. Data in panels D and E are means of three independent determinations, in which at least 100 fiber measurements were performed. Error bars represent standard deviations. (F) Efficacy of HLTF knockdown by siRNA and HLTF expression. Total cell extracts were prepared from aliquots of samples used for preparing DNA fibers. The efficiency of HLTF knockdown and expression of wild-type, ATPase, RING, and RING/ATPase double-mutant 6MYC-HLTF proteins were confirmed by Western blotting using anti-HLTF antibody. As a loading control, antitubulin antibody was used.

FIG. 6.

Model for template switch damage bypass by HLTF-dependent fork reversal and strand invasion. Stalling of replication at an unrepaired DNA lesion (X) can lead to unresolved fork structures as well as gaps in the newly synthesized daughter strand opposite the lesion. Unresolved fork structures can particularly be formed if replication cannot be reinitiated downstream of a fork-blocking lesion and cryptic origins of replication cannot be activated at intradamage DNA fragments. The filling in of gaps and resolving fork structures can require postreplicative repair mechanisms. (I) Damage bypass by HLTF-dependent fork reversal. (A) When the lesion is located on the leading strand template, the leading and lagging strand synthesis can become uncoupled, and synthesis on the lagging strand can continue way past the blocked nascent leading strand. (B) The nascent strands then unwind from their respective templates and anneal with one another, and the parental strands also reanneal, which can be catalyzed by HLTF. The overall outcome of these reactions is the regression of the replication fork to form a four-way Holliday junction. (C) Following that, the sequences complementary to the damaged region are synthesized on the nascent leading strand using the nascent lagging strand as the template. (D) The regressed fork is then reversed, and synthesis resumes beyond the point of the lesion. (II) Damage bypass by strand invasion. The 3′ end of the newly synthesized gapped DNA invades the homologous region of the sister chromatid and uses its newly synthesized strand as a template for DNA synthesis.