Zhihao Zhuang

Regulation of polymerase exchange between Polη and Polδ by monoubiquitination of PCNA and the movement of DNA polymerase holoenzyme

To ensure efficient and timely replication of genomic DNA, organisms in all three kingdoms of life possess specialized translesion DNA synthesis (TLS) polymerases (Pols) that tolerate various types of DNA lesions. It has been proposed that an exchange between the replicative DNA Pol and the TLS Pol at the site of DNA damage enables lesion bypass to occur. However, to date the molecular mechanism underlying this process is not fully understood. In this study, we demonstrated in a reconstituted system that the exchange of Saccharomyces cerevisiae Polδ with Polη requires both the stalling of the holoenzyme and the monoubiquitination of proliferating cell nuclear antigen (PCNA). A moving Polδ holoenzyme is refractory to the incoming Polη. Furthermore, we showed that the Polη C-terminal PCNA-interacting protein motif is required for the exchange process. We also demonstrated that the second exchange step to bring back Polδ is prohibited when Lys-164 of PCNA is monoubiquitinated. Thus the removal of the ubiquitin moiety from PCNA is likely required for the reverse exchange step after the lesion bypass synthesis by Polη.

Chemically ubiquitylated PCNA as a probe for eukaryotic translesion DNA synthesis

The rapid growth in ubiquitin biology requires facile chemical approaches for protein ubiquitylation that can overcome the common problem of low yield faced by the enzymatic reaction catalyzed by ubiquitin ligases. We report a chemical approach for monoubiquitylation and SUMOylation of PCNA through disulfide exchange and intein chemistry. We used the chemically ubiquitylated and SUMOylated PCNAs in studying translesion DNA synthesis and revealed a surprising degree of flexibility of the ubiquitin modification.

Structure, Function, and Mechanism of the Phenylacetate Pathway Hot Dog-fold Thioesterase PaaI

The structure and biochemical function of the hot dog-fold thioesterase PaaI operative in the aerobic phenylacetate degradation pathway are examined. PaaI showed modest activity with phenylacetyl-coenzyme A, suggestive of a role in coenzyme A release from this pathway intermediate in the event of limiting downstream pathway enzymes. Minimal activity was observed with aliphatic acyl-coenzyme A thioesters, which ruled out PaaI function in the lower phenylacetate pathway. PaaI was most active with ring-hydroxylated phenylacetyl-coenzyme A thioesters. The x-ray crystal structure of the Escherichia coli thioesterase is reported and analyzed to define the structural basis of substrate recognition and catalysis. The contributions of catalytic and substrate binding residues, thus, identified were examined through steady-state kinetic analysis of site-directed mutant proteins.

A selective USP1–UAF1 inhibitor links deubiquitination to DNA damage responses

Protein ubiquitination and deubiquitination are central to the control of a large number of cellular pathways and signaling networks in eukaryotes. Although the essential roles of ubiquitination have been established in the eukaryotic DNA damage response, the deubiquitination process remains poorly defined. Chemical probes that perturb the activity of deubiquitinases (DUBs) are needed to characterize the cellular function of deubiquitination. Here we report ML323 (2), a highly potent inhibitor of the USP1-UAF1 deubiquitinase complex with excellent selectivity against human DUBs, deSUMOylase, deneddylase and unrelated proteases. Using ML323, we interrogated deubiquitination in the cellular response to UV- and cisplatin-induced DNA damage and revealed new insights into the requirement of deubiquitination in the DNA translesion synthesis and Fanconi anemia pathways. Moreover, ML323 potentiates cisplatin cytotoxicity in non-small cell lung cancer and osteosarcoma cells. Our findings point to USP1-UAF1 as a key regulator of the DNA damage response and a target for overcoming resistance to the platinum-based anticancer drugs.

The YbgC protein encoded by the ybgC gene of the tol-pal gene cluster of Haemophilus influenzae catalyzes acyl-coenzyme A thioester hydrolysis

This paper examines the catalytic function of the protein YbgC, encoded by the ybgC gene of the tol‐pal gene cluster in Haemophilus influenzae. The YbgC protein, a homologue of the Pseudomonas sp. strain CBS3 4‐hydroxybenzoyl‐coenzyme A thioesterase, conserves the active site Asp residue associated with thioesterase activity. The H. influenzae ybgC gene was cloned and overexpressed in Escherichia coli. The recombinant protein was purified and tested for thioesterase activity towards acyl‐CoA and acyl‐N‐acetylcysteamine thioesters. The YbgC protein catalyzes the hydrolysis of short chain aliphatic acyl‐CoA thioesters, while the D18N YbgC mutant protein (prepared to serve as a control) does not.

Characterization of human Spartan/C1orf124, an ubiquitin-PCNA interacting regulator of DNA damage tolerance

Unrepaired DNA damage may arrest ongoing replication forks, potentially resulting in fork collapse, increased mutagenesis and genomic instability. Replication through DNA lesions depends on mono- and polyubiquitylation of proliferating cell nuclear antigen (PCNA), which enable translesion synthesis (TLS) and template switching, respectively. A proper replication fork rescue is ensured by the dynamic ubiquitylation and deubiquitylation of PCNA; however, as yet, little is known about its regulation. Here, we show that human Spartan/C1orf124 protein provides a higher cellular level of ubiquitylated-PCNA by which it regulates the choice of DNA damage tolerance pathways. We find that Spartan is recruited to sites of replication stress, a process that depends on its PCNA- and ubiquitin-interacting domains and the RAD18 PCNA ubiquitin ligase. Preferential association of Spartan with ubiquitin-modified PCNA protects against PCNA deubiquitylation by ubiquitin-specific protease 1 and facilitates the access of a TLS polymerase to the replication fork. In concert, depletion of Spartan leads to increased sensitivity to DNA damaging agents and causes elevated levels of sister chromatid exchanges. We propose that Spartan promotes genomic stability by regulating the choice of rescue of stalled replication fork, whose mechanism includes its interaction with ubiquitin-conjugated PCNA and protection against PCNA deubiquitylation.

The structure of 4-hydroxybenzoyl-CoA thioesterase from arthrobacter sp. strain SU.

The 4-chlorobenzoyl-CoA dehalogenation pathway in certain Arthrobacter and Pseudomonas bacterial species contains three enzymes: a ligase, a dehalogenase, and a thioesterase. Here we describe the high resolution x-ray crystallographic structure of the 4-hydroxybenzoyl-CoA thioesterase from Arthrobacter sp. strain SU. The tetrameric enzyme is a dimer of dimers with each subunit adopting the so-called “hot dog fold” composed of six strands of anti-parallel β-sheet flanked on one side by a rather long α-helix. The dimers come together to form the tetramer with their α-helices facing outwards. This quaternary structure is in sharp contrast to that previously observed for the 4-hydroxybenzoyl-CoA thioesterase from Pseudomonas species strain CBS-3, whereby the dimers forming the tetramer pack with their α-helices projecting toward the interfacial region. In the Arthrobacter thioesterase, each of the four active sites is formed by three of the subunits of the tetramer. On the basis of both structural and kinetic data, it appears that Glu73 is the active site base in the Arthrobacter thioesterase. Remarkably, this residue is located on the opposite side of the substrate-binding pocket compared with that observed for the Pseudomonas enzyme. Although these two bacterial thioesterases demonstrate equivalent catalytic efficiencies, substrate specificities, and metabolic functions, their quaternary structures, CoA-binding sites, and catalytic platforms are decidedly different.

Solution X-ray scattering combined with computational modeling reveals multiple conformations of covalently bound ubiquitin on PCNA

PCNA ubiquitination in response to DNA damage leads to the recruitment of specialized translesion polymerases to the damage locus. This constitutes one of the initial steps in translesion synthesis (TLS)—a critical pathway for cell survival and for maintenance of genome stability. The recent crystal structure of ubiquitinated PCNA (Ub–PCNA) sheds light on the mode of association between the two proteins but also revealed that paradoxically, the ubiquitin surface engaged in PCNA interactions was the same as the surface implicated in translesion polymerase binding. This finding implied a degree of flexibility inherent in the Ub–PCNA complex that would allow it to transition into a conformation competent to bind the TLS polymerase. To address the issue of segmental flexibility, we combined multiscale computational modeling and small angle X-ray scattering. This combined strategy revealed alternative positions for ubiquitin to reside on the surface of the PCNA homotrimer, distinct from the position identified in the crystal structure. Two mutations originally identified in genetic screens and known to interfere with TLS are positioned directly beneath the bound ubiquitin in the alternative models. These computationally derived positions, in an ensemble with the crystallographic and flexible positions, provided the best fit to the solution scattering, indicating that ubiquitin dynamically associated with PCNA and is capable of transitioning between a few discrete sites on the PCNA surface. The finding of new docking sites and the positional equilibrium of PCNA–Ub occurring in solution provide unexpected insight into previously unexplained biological observations.

Ubiquitination of PCNA and Its Essential Role in Eukaryotic Translesion Synthesis

Ubiquitin and ubiquitin-like proteins (Ubls) are now at the center stage of molecular and cell biology because of their diverse functions in many fundamentally important cellular processes. Besides the celebrated role of ubiquitin in the 26S proteasome-mediated protein degradation pathway, the non-proteolytic functions of ubiquitin are being uncovered at a fast pace. The prominent examples include membrane trafficking, innate immunity, kinase signaling, chromatin dynamics and DNA damage response. Researchers in the area of DNA damage response have witnessed rapid progress within the past decade, largely stimulated by the seminal findings that ubiquitination and SUMOylation of a key DNA replication/repair protein, proliferating cell nuclear antigen (PCNA), controls precisely how eukaryotic cells respond to different types of DNA damage, and how cellular DNA damage repair or tolerance pathways are selected to cope with damage in the DNA genome. Here, we will review the recent findings on translesion synthesis (TLS) and its regulation by PCNA ubiquitination in eukaryotes. We will discuss two prevalent models, i.e., the postreplicative gap-filling and the polymerase switch, which have been invoked to account for eukaryotic cells’ ability to overcome DNA damage associated replication blockade through TLS. Results from both in vitro reconstitution and from genetic systems will be discussed. We will also summarize the recent findings revealing the crosstalk between two major human DNA damage response pathways (the TLS and the Fanconi anemia pathways), and the ATR and ATM-independent regulation of PCNA ubiquitination. Lastly, new methods of preparing ubiquitinated PCNA will be reviewed. The availability of milligram levels of ubiquitinated PCNA will help our understanding of the molecular details in eukaryotic TLS.

A Noncanonical Cysteine Protease USP1 Is Activated through Active Site Modulation by USP1-Associated Factor 1

Ubiquitin-specific proteases (USPs) constitute the largest family of the human deubiquitinating enzymes. USP1 belongs to the cysteine protease family and contains a catalytic triad comprised of C90, H593, and D751. Notably, the catalytic activity of USP1 is stimulated through the formation of a tight complex with a WD40 repeat protein UAF1 (USP1-associated factor 1). Our kinetic analyses revealed a general base catalysis in USP1/UAF1, in contrast to an ion-pair mechanism as demonstrated for papain and cathepsin. The pK(a) value of the catalytic cysteine was determined to be 8.67 ± 0.07 in a pH-dependent inactivation study of USP1/UAF1 by iodoacetamide. A normal solvent kinetic isotope effect of 2.8 for k(cat) and 3.0 for k(cat)/K(m) was observed in the USP1/UAF1-catalyzed hydrolysis of ubiquitin-AMC substrate. Moreover, proton inventory analysis supported the transfer of a single solvent-derived proton in the transition state. Our study also revealed the molecular basis for the activation of USP1 by UAF1. Although the pK(a) of the catalytic cysteine in USP1 and USP1/UAF1 was almost identical, the pK(a) of the catalytic histidine in USP1/UAF1 was 0.43 pH unit lower than that in USP1, which facilitates general base catalysis at a neutral pH and contributes to the elevated catalytic efficiency. We ruled out that the higher catalytic efficiency is due to a tighter binding of ubiquitin. Our results support a regulatory mechanism in which UAF1 activates USP1 by modulating its active site conformation. This finding has a general implication for the regulation of USPs that form complex with partner proteins.