Jerard Hurwitz

Structure of the C-Terminal Region of p21WAF1/CIP1 Complexed with Human PCNA

The crystal structure of the human DNA polymerase delta processivity factor PCNA (proliferating cell nuclear antigen) complexed with a 22 residue peptide derived from the C-terminus of the cell-cycle checkpoint protein p21(WAF1/CIP1) has been determined at 2.6 angstrom resolution. p21 binds to PCNA in a 1:1 stoichiometry with an extensive array of interactions that include the formation of a beta sheet with the interdomain connector loop of PCNA. An intact trimeric ring is maintained in the structure of the p21-PCNA complex, with a central hole available for DNA interaction. The ability of p21 to inhibit the action of PCNA is therefore likely to be due to its masking of elements on PCNA that are required for the binding of other components of the polymerase assembly.

RNA Helicase A Mediates Association of CBP with RNA Polymerase II

The coactivator CBP has been proposed to stimulate the expression of certain signal-dependent genes via its association with RNA polymerase II complexes. Here we show that complex formation between CBP and RNA polymerase II requires RNA helicase A (RHA), a nuclear DNA/RNA helicase that is related to the Drosophila male dosage compensation factor mle. In transient transfection assays, RHA was found to cooperate with CBP in mediating target gene activation via the CAMP responsive factor CREB. As a mutation in RHA that compromised its helicase activity correspondingly reduced CREB-dependent transcription, we propose that RHA may induce local changes in chromatin structure that promote engagement of the transcriptional apparatus on signal responsive promoters.

Loading of the human 9-1-1 checkpoint complex onto DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro

The human DNA damage sensors, Rad17-replication factor C (Rad17-RFC) and the Rad9-Rad1-Hus1 (9-1-1) checkpoint complex, are thought to be involved in the early steps of the DNA damage checkpoint response. Rad17-RFC and the 9-1-1 complex have been shown to be structurally similar to the replication factors, RFC clamp loader and proliferating cell nuclear antigen polymerase clamp, respectively. Here, we demonstrate functional similarities between the replication and checkpoint clamp loader/DNA clamp pairs. When all eight subunits of the two checkpoint complexes are coexpressed in insect cells, a stable Rad17-RFC/9-1-1 checkpoint supercomplex forms in vivo and is readily purified. The two individually purified checkpoint complexes also form a supercomplex in vitro, which depends on ATP and is mediated by interactions between Rad17 and Rad9. Rad17-RFC binds to nicked circular, gapped, and primed DNA and recruits the 9-1-1 complex in an ATP-dependent manner. Electron microscopic analyses of the reaction products indicate that the 9-1-1 ring is clamped around the DNA.

Binding and unwinding—How T antigen engages the SV40 origin of DNA replication

The development of a cell-free system that catalyzes SV40 DNA replication in vitro coupled with the use of expression systems that overproduce T antigen has allowed detailed analysis of the individual steps carried out by T antigen prior to initiation of DNA synthesis. This review will focus on events preceding initiation DNA synthesis

Purification and characterization of human DNA damage checkpoint Rad complexes

Checkpoint Rad proteins function early in the DNA damage checkpoint signaling cascade to arrest cell cycle progression in response to DNA damage. This checkpoint ensures the transmission of an intact genetic complement to daughter cells. To learn about the damage sensor function of the human checkpoint Rad proteins, we purified a heteropentameric complex composed of hRad17-RFCp36-RFCp37-RFCp38-RFCp40 (hRad17-RFC) and a heterotrimeric complex composed of hRad9-hHus1-hRad1 (checkpoint 9-1-1 complex). hRad17-RFC binds to DNA, with a preference for primed DNA and possesses weak ATPase activity that is stimulated by primed DNA and single-stranded DNA. hRad17-RFC forms a complex with the 9-1-1 heterotrimer reminiscent of the replication factor C/proliferating cell nuclear antigen clamp loader/sliding clamp complex of the replication machinery. These findings constitute biochemical support for models regarding the roles of checkpoint Rads as damage sensors in the DNA damage checkpoint response of human cells.

Targeting of human DNA polymerase ι to the replication machinery via interaction with PCNA

Human DNA polymerase ι (hPolι) promotes translesion synthesis by inserting nucleotides opposite highly distorting or noninstructional DNA lesions. Here, we provide evidence for the physical interaction of hPolι with proliferating cell nuclear antigen (PCNA), and show that PCNA, together with replication factor C (RFC) and replication protein A (RPA), stimulates the DNA synthetic activity of hPolι. In the presence of these protein factors, on undamaged DNA, the efficiency (Vmax/Km) of correct nucleotide incorporation by hPolι is increased ≈80–150-fold, and this increase in efficiency results from a reduction in the apparent Km for the nucleotide. PCNA, RFC, and RPA also stimulate nucleotide incorporation opposite the 3′-T of the (6) thymine–thymine (T-T) photoproduct and opposite an abasic site. The interaction of hPolι with PCNA implies that the targeting of this polymerase to the replication machinery stalled at a lesion site is achieved via this association.

Multiple competition reactions for RPA order the assembly of the DNA polymerase delta holoenzyme.

Processive extension of DNA in eukaryotes requires three factors to coordinate their actions. First, DNA polymerase α‐primase synthesizes the primed site. Then replication factor C loads a proliferating cell nuclear antigen (PCNA) clamp onto the primer. Following this, DNA polymerase δ assembles with PCNA for processive extension. This report shows that these proteins each bind the primed site tightly and trade places in a highly coordinated fashion such that the primer terminus is never left free of protein. Replication protein A (RPA), the single‐stranded DNA‐binding protein, forms a common touchpoint for each of these proteins and they compete with one another for it. Thus these protein exchanges are driven by competition‐based protein switches in which two proteins vie for contact with RPA.

Clamp loading, unloading and intrinsic stability of the PCNA, β and gp45 sliding clamps of human, E. coli and T4 replicases

Background: The high speed and processivity of replicative DNA polymerases reside in a processivity factor which has been shown to be a ring‐shaped protein. This protein (‘sliding clamp’) encircles DNA and tethers the catalytic unit to the template. Although in eukaryotic, prokaryotic and bacteriophage‐T4 systems, the processivity factors are ring‐shaped, they assume different oligomeric states. The Escherichia coli clamp (the β subunit) is active as a dimer while the eukaryotic and T4 phage clamps (PCNA and gp45, respectively) are active as trimers. The clamp can not assemble itself on DNA. Instead, a protein complex known as a clamp loader utilizes ATP to assemble the ring around the primer‐template. This study compares properties of the human PCNA clamp with those of E. coli and T4 phage.

Human SHPRH is a ubiquitin ligase for Mms2–Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen

Human SHPRH gene is located at the 6q24 chromosomal region, and loss of heterozygosity in this region is seen in a wide variety of cancers. SHPRH is a member of the SWI/SNF family of ATPases/helicases, and it possesses a C3HC4 RING motif characteristic of ubiquitin ligase proteins. In both of these features, SHPRH resembles the yeast Rad5 protein, which, together with Mms2–Ubc13, promotes replication through DNA lesions via an error-free postreplicational repair pathway. Genetic evidence in yeast has indicated a role for Rad5 as a ubiquitin ligase in mediating the Mms2–Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen. Here we show that SHPRH is a functional homolog of Rad5. Similar to Rad5, SHPRH physically interacts with the Rad6–Rad18 and Mms2–Ubc13 complexes, and we show that SHPRH protein is a ubiquitin ligase indispensable for Mms2–Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen. Based on these observations, we predict a role for SHPRH in promoting error-free replication through DNA lesions. Such a role for SHPRH is consistent with the observation that this gene is mutated in a number of cancer cell lines, including those from melanomas and ovarian cancers, which raises the strong possibility that SHPRH function is an important deterrent to mutagenesis and carcinogenesis in humans.

Protein–PCNA interactions: a DNA-scanning mechanism?

Proliferating-cell nuclear antigen (PCNA) plays an essential role in nucleic-acid metabolism in all eukaryotes. The PCNA protein interacts with a large number of proteins. These proteins can be divided into two groups: the first contains proteins that have a known enzymatic activity; the second contains regulatory proteins that are involved in cell-cycle progression, checkpoint control and cellular differentiation. Interestingly, all of the enzymes known to interact with PCNA either recognize specific structures on DNA or have limited DNA-sequence specificity. Proteins that have low sequence specificities could utilize PCNA as an adapter in order to interact with their DNA substrates.