Human HLTF functions as a ubiquitin ligase for proliferating cell nuclear antigen polyubiquitination

Abstract

Human helicase-like transcription factor (HLTF) is frequently inactivated in colorectal and gastric cancers. Here, we show that HLTF is a functional homologue of yeast Rad5 that promotes error-free replication through DNA lesions. HLTF and Rad5 share the same unique structural features, including a RING domain embedded within a SWI/SNF helicase domain and an HIRAN domain. We find that inactivation of HLTF renders human cells sensitive to UV and other DNA-damaging agents and that HLTF complements the UV sensitivity of a rad5⌬ yeast strain. Also, similar to Rad5, HLTF physically interacts with the Rad6 -Rad18 and Mms2-Ubc13 ubiquitin-conjugating enzyme complexes and promotes the Lys-63-linked polyubiquitination of proliferating cell nuclear antigen at its Lys-164 residue. A requirement of HLTF for error-free postreplication repair of damaged DNA is in keeping with its cancersuppression role.yeast Rad5 ͉ damage bypass ͉ K63 polyubiquitination ͉ tumor suppressor L esions in DNA impose a block to synthesis by the replicative polymerases (Pols), and unless replication is rescued by the timely action of lesion bypass processes, stalled replication forks can collapse, leading to genomic instability. In eukaryotes the Rad6-Rad18 enzyme complex regulates lesion bypass processes that ensure the completion of replication. Rad6, a ubiquitinconjugating enzyme, forms a tight complex with Rad18, a RING-finger type ubiquitin ligase that binds DNA (1, 2), and in cells treated with DNA-damaging agents, Rad6-Rad18 monoubiquitinates proliferating cell nuclear antigen (PCNA), a DNA Pol sliding clamp that is a key component of the replication machinery (3). Ubiquitination at the Lys-164 residue of PCNA and its subsequent polyubiquitination serves as a molecular switch between various DNA damage bypass processes (3-5).In the yeast Saccharomyces cerevisiae, Rad6-Rad18 governs at least three alternative pathways for promoting replication through DNA lesions (6). Two pathways activated by PCNA monoubiquitination are carried out by specialized translesion synthesis (TLS) DNA Pols, such as Pol and Pol , which are able to copy DNA directly from the damaged template on an error-free or error-prone way (6). The third pathway called postreplication repair (PRR), however, is activated by PCNA polyubiquitination and operates by template switching using the information of the undamaged newly synthesized nascent strand on the sister duplex for DNA synthesis across damaged DNA (7-10). The PRR pathway depends on the Rad5, Mms2, and Ubc13 proteins and promotes error-free replication through DNA lesions. Recently, we have shown that yeast Rad5 has a DNA helicase activity that is specialized for replication fork regression, as Rad5 can concertedly unwind and anneal the nascent and the parental strands of the fork without exposing any single-stranded regions (7). This Rad5 activity would ensure damage bypass by promoting replication fork regression where the newly synthesized DNA strand of the sister duplex can be used as a template. In addition to its r...

SHPRH regulates rRNA transcription by recognizing the histone code in an mTOR-dependent manner

“…SHPRH and HLTF, homologs of yeast Rad5, were previously identified as E3 ubiquitin ligases that catalyze the polyubiquitylation of PCNA and participate in methyl methane sulfonate (MMS)-induced DNA damage repair (26)(27)(28). In contrast to yeast in which Rad5-deficiency causes high sensitivity to DNA-damaging agents, silencing expression of SHPRH or HLTF in human cells or knockouts of SHPRH and HLTF in mice showed mild or no severe sensitivity to DNA damaging agents, respectively (29)(30)(31) (Fig.…”

“…In contrast to yeast Rad5 or human HLTF, SHPRH has a PHD (22,28), which is generally known to interact with specifically modified histone H3 (Fig. 3A) (37)(38)(39).…”

“…RAD5 and its mammalian homolog SHPRH polyubiquitylate proliferating cell nuclear antigen (PCNA) to promote DNA damage bypass via an uncharacterized recombination-dependent pathway (24,25). Furthermore, there are two RAD5 homologs in mammals, which suggests that functions other than DNA damage bypass might have been developed during evolution (26)(27)(28).…”

BRCA1promotes the ubiquitination of PCNA and recruitment of translesion polymerases in response to replication blockade

“…Both branches of the DDT pathways appear to be more complex in mammals. For example, two Saccharomyces cerevisiae Rad5 homologs have been identified in mammals, HLTF and SHPRH (19)(20)(21)(22). Both can bind the E2 heterodimer, UBC13 and MMS2, and promote the polyubiquitination of PCNA in vitro and in vivo.…”

HIV-1 Vpr degrades the HLTF DNA translocase in T cells and macrophages

“…HLTF is an enzyme that stabilizes and repairs stalled replication forks and controls G2/M transition (49,59,60). Considering HLTF activities led us to investigate its role in Vpr-mediated G2 arrest.…”