No abstract

DNA repair proteins conduct a genome-wide search to detect and repair sites of DNA damage wherever they occur. Human alkyladenine DNA glycosylase (AAG) is responsible for recognizing a variety of base lesions, including alkylated and deaminated purines, and initiating their repair via the base excision repair pathway. We have investigated the mechanism by which AAG locates sites of damage using an oligonucleotide substrate containing two sites of DNA damage. This substrate was designed so that AAG randomly binds to either of the two lesions. AAG-catalyzed base excision creates a repair intermediate and the subsequent partitioning between dissociation and diffusion to the second site can be quantified from the rates of formation of the different products. Our results demonstrate that AAG has the ability to slide for short distances along DNA at physiological salt concentrations. The processivity of AAG decreases with increasing ionic strength to become fully distributive at high ionic strength, suggesting that electrostatic interactions between the negatively charged DNA and the positively charged DNA binding surface are important for nonspecific DNA binding. Although the amino terminus of the protein is dispensable for glycosylase activity at a single site, we find that deletion of the amino terminal 80 amino acids significantly decreases the processivity of AAG. These observations support the idea that diffusion on undamaged DNA contributes to the search for sites of DNA damage.Although DNA is remarkably stable, it is nevertheless susceptible to spontaneous damage via reactions with cellular metabolites and environmental mutagens. Chemical reactions that alter the structure of the nucleobases within DNA are most commonly recognized and repaired by the base excision repair (BER) 1 pathway. Some base lesions can block DNA replication and transcription with cytotoxic effects, and many more alter the base pairing properties so that replication leads to mispairing and mutation. The base excision repair pathway is initiated by a DNA repair glycosylase that must locate the site of damage within the genome. Once a damaged base is located, the glycosylase flips out the damaged nucleotide and catalyzes the hydrolysis of the N-glycosidic bond to release the lesioned base. The subsequent actions of an endonuclease, abasic site lyase, DNA polymerase and DNA ligase are required to complete the repair pathway.It is estimated that ∼10,000 base lesions are formed in a typical human cell every day and that the vast majority of these are correctly repaired by BER or other DNA repair pathways (4). On the one hand, this is a large number of potential mutagenic events that must be corrected. On the other hand, these lesions are very rare considering the size of the human diploid genome † This work was supported by a grant from the NIH to P.O. (CA122254).* Address correspondence to P.O. at the Department of Biological Chemistry, University of Michigan Medical School, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0606. E-mai...

Replicative polymerases (pols) cannot accommodate damaged template bases, and these pols stall when such offenses are encountered during S phase. Rather than repairing the damaged base, replication past it may proceed via one of two DNA damage tolerance (DDT) pathways, allowing replicative DNA synthesis to resume. In translesion DNA synthesis (TLS), a specialized TLS pol is recruited to catalyze stable, yet often erroneous, nucleotide incorporation opposite damaged template bases. In template switching, the newly synthesized sister strand is used as a damage-free template to synthesize past the lesion. In eukaryotes, both pathways are regulated by the conjugation of ubiquitin to the PCNA sliding clamp by distinct E2/E3 pairs. Whereas monoubiquitination by Rad6/Rad18 mediates TLS, extension of this ubiquitin to a polyubiquitin chain by Ubc13-Mms2/Rad5 routes DDT to the template switching pathway. In this review, we focus on the monoubiquitination of PCNA by Rad6/Rad18 and summarize the current knowledge of how this process is regulated.

Spontaneous DNA damage occurs throughout the genome, requiring that DNA repair enzymes search each nucleotide every cell cycle. This search is postulated to be more efficient if the enzyme can diffuse along the DNA, but our understanding of this process is incomplete. A key distinction between mechanisms of diffusion is whether the protein maintains continuous contact (sliding) or whether it undergoes microscopic dissociation (hopping). We describe a simple chemical assay to detect the ability of a DNA modifying enzyme to hop and have applied it to human alkyladenine DNA glycosylase (AAG), a monomeric enzyme that initiates repair of alkylated and deaminated purine bases. Our results indicate that AAG uses hopping to effectively search both strands of a DNA duplex in a single binding encounter. This raised the possibility that AAG might be capable of circumnavigating blocks such as tightly bound proteins. We tested this hypothesis by binding an EcoRI endonuclease dimer between two sites of DNA damage and measuring the ability of AAG to act at both damaged sites in a single binding encounter. Remarkably, AAG bypasses this roadblock in ∼50% of the binding events. We infer that AAG makes significant excursions from the surface of the DNA, allowing reorientation between strands and the bypass of a bound protein. This has important biological implications for the search for DNA damage because eukaryotic DNA is replete with proteins and only transiently accessible.The human base excision DNA repair pathway repairs ∼10,000 lesions per cell per day (1). This is a daunting task because these relatively rare lesions must be located from among ∼12,000,000,000 normal nucleotides in the genome. Almost a dozen different human DNA repair glycosylases continuously and independently search the genome for a wide variety of oxidized or alkylated bases. Once a damaged nucleotide has been located, the glycosylase catalyzes the hydrolysis of the N-glycosidic bond to release the damaged base and create an abasic site. This abasic site is further processed to restore the correct DNA sequence using the opposing nucleotide as a template. There is considerable in vitro evidence that glycosylases use thermally-driven linear diffusion to efficiently search for sites of damage, whereby the enzyme diffuses along DNA in a non-directional manner, searching many adjacent sites within a single binding event (2-8). The biological importance of linear diffusion has been confirmed by the findings that mutants of T4 pyrimidine dimer glycosylase and EcoRV endonuclease that are deficient in linear diffusion have decreased activity in vivo (9-11).The task of locating specific sites within the genome is central to DNA repair and to many other nuclear processes such as DNA replication and transcription (12)(13)(14). Two distinct mechanisms are recognized for diffusion along DNA and they are commonly referred to as [13][14][15][16][17][18][19][20][21][22]. As illustrated in Scheme 1, sliding involves continuous contact between the protein and the D...

To achieve the high degree of processivity required for DNA replication, DNA polymerases associate with ring-shaped sliding clamps that encircle the template DNA and slide freely along it. The closed circular structure of sliding clamps necessitates an enzyme-catalyzed mechanism, which not only opens them for assembly and closes them around DNA, but specifically targets them to sites where DNA synthesis is initiated and orients them correctly for replication. Such a feat is performed by multisubunit complexes known as clamp loaders, which use ATP to open sliding clamp rings and place them around the 3′ end of primer–template (PT) junctions. Here we discuss the structure and composition of sliding clamps and clamp loaders from the three domains of life as well as T4 bacteriophage, and provide our current understanding of the clamp-loading process.

DNA replication requires the sliding clamp, a ring-shaped protein complex that encircles DNA, where it acts as an essential cofactor for DNA polymerases and other proteins. The sliding clamp needs to be opened and installed onto DNA by a clamp loader ATPase of the AAA+ family. The human clamp loader replication factor C (RFC) and sliding clamp proliferating cell nuclear antigen (PCNA) are both essential and play critical roles in several diseases. Despite decades of study, no structure of human RFC has been resolved. Here, we report the structure of human RFC bound to PCNA by cryogenic electron microscopy to an overall resolution of ∼3.4 Å. The active sites of RFC are fully bound to adenosine 5′-triphosphate (ATP) analogs, which is expected to induce opening of the sliding clamp. However, we observe the complex in a conformation before PCNA opening, with the clamp loader ATPase modules forming an overtwisted spiral that is incapable of binding DNA or hydrolyzing ATP. The autoinhibited conformation observed here has many similarities to a previous yeast RFC:PCNA crystal structure, suggesting that eukaryotic clamp loaders adopt a similar autoinhibited state early on in clamp loading. Our results point to a “limited change/induced fit” mechanism in which the clamp first opens, followed by DNA binding, inducing opening of the loader to release autoinhibition. The proposed change from an overtwisted to an active conformation reveals an additional regulatory mechanism for AAA+ ATPases. Finally, our structural analysis of disease mutations leads to a mechanistic explanation for the role of RFC in human health.

In eukaryotes, DNA polymerase δ (pol δ) is responsible for replicating the lagging strand template and anchors to the proliferating cell nuclear antigen (PCNA) sliding clamp to form a holoenzyme. The stability of this complex is integral to every aspect of lagging strand replication. Most of our understanding comes from Saccharomyces cerevisae where the extreme stability of the pol δ holoenzyme ensures that every nucleobase within an Okazaki fragment is faithfully duplicated before dissociation but also necessitates an active displacement mechanism for polymerase recycling and exchange. However, the stability of the human pol δ holoenzyme is unknown. We designed unique kinetic assays to analyze the processivity and stability of the pol δ holoenzyme. Surprisingly, the results indicate that human pol δ maintains a loose association with PCNA while replicating DNA. Such behavior has profound implications on Okazaki fragment synthesis in humans as it limits the processivity of pol δ on undamaged DNA and promotes the rapid dissociation of pol δ from PCNA on stalling at a DNA lesion.lagging strand | stability | PCNA | DNA polymerase delta | translesion DNA synthesis D uring S-phase of the cell cycle, genomic DNA must be faithfully copied in a short period. Replicative DNA polymerases (pols) alone are distributive and must anchor to ringshaped sliding clamps to achieve the high degree of processivity required for efficient DNA replication. The highly conserved toroidal structure of sliding clamps has a central cavity large enough to encircle double-stranded DNA (dsDNA) and slide freely along it. Thus, such an association effectively tethers the pol to DNA, substantially increasing the extent of continuous replication. The eukaryotic sliding clamp, proliferating cell nuclear antigen (PCNA), is trimer of identical subunits aligned head-to-tail, forming a ring with two structurally distinct faces. Each subunit consists of two independent domains connected by an interdomain connecting loop (IDCL). The "front" face of the homotrimeric PCNA ring contains all IDCLs and is a platform for interaction with the eukaryotic replicative pols, e and δ, which are responsible for the faithful replication of the leading and lagging strands, respectively (1, 2). Specifically, the well-conserved PCNA-interacting peptide (PIP) box within replicative pols makes extensive contact with an IDCL of PCNA and displays conserved residues that "plug" into the proximal hydrophobic patches. The amino acid sequence of a canonical PIP box is QXXhXXaa, where X represents any amino acid, h is a hydrophobic residue (usually L, I, or M), and a is an aromatic residue (usually F or Y) (3).Unlike the leading strand, the lagging strand is synthesized discontinuously in short Okazaki fragments that are processed and ligated together to form a continuous strand (4). In eukaryotes, each Okazaki fragment is initiated by the bifunctional DNA pol α/primase complex that lays down cRNA/DNA hybrid primers every 100-250 nucleotides (nt) on the exposed template for the l...

In most organisms, clamp loaders catalyze both the loading of sliding clamps onto DNA and their removal. How these opposing activities are regulated during assembly of the DNA polymerase holoenzyme remains unknown. By utilizing FRET to monitor protein-DNA interactions, we examined assembly of the human holoenzyme. The results indicate that assembly proceeds in a stepwise manner. The clamp loader (RFC) loads a sliding clamp (PCNA) onto a primer/template junction but remains transiently bound to the DNA. Unable to slide away, PCNA re-engages with RFC and is unloaded. In the presence of polymerase (polδ), loaded PCNA is captured from DNA-bound RFC which subsequently dissociates, leaving behind the holoenzyme. These studies suggest that the unloading activity of RFC maximizes the utilization of PCNA by inhibiting the build-up of free PCNA on DNA in the absence of polymerase and recycling limited PCNA to keep up with ongoing replication.DOI: http://dx.doi.org/10.7554/eLife.00278.001