Stepwise requirements for polymerases δ and θ in theta-mediated end joining.

Timely repair of chromosomal double-strand breaks is required for genome integrity and cellular viability. The polymerase theta-mediated end joining pathway has an important role in resolving these breaks and is essential in cancers defective in other DNA repair pathways, thus making it an emerging therapeutic target1. It requires annealing of 2-6 nucleotides of complementary sequence, microhomologies, that are adjacent to the broken ends, followed by initiation of end-bridging DNA synthesis by polymerase θ. However, the other pathway steps remain inadequately defined, and the enzymes required for them are unknown. Here we demonstrate requirements for exonucleolytic digestion of unpaired 3' tails before polymerase θ can initiate synthesis, then a switch to a more accurate, processive and strand-displacing polymerase to complete repair. We show the replicative polymerase, polymerase δ, is required for both steps; its 3' to 5' exonuclease activity for flap trimming, then its polymerase activity for extension and completion of repair. The enzymatic steps that are essential and specific to this pathway are mediated by two separate, sequential engagements of the two polymerases. The requisite coupling of these steps together is likely to be facilitated by physical association of the two polymerases. This pairing of polymerase δ with a polymerase capable of end-bridging synthesis, polymerase θ, may help to explain why the normally high-fidelity polymerase δ participates in genome destabilizing processes such as mitotic DNA synthesis2 and microhomology-mediated break-induced replication3.

Replication protein A dynamically re-organizes on primer/template junctions to permit DNA polymerase δ holoenzyme assembly and initiation of DNA synthesis.

DNA polymerase δ (pol δ) holoenzymes, comprised of pol δ and the processivity sliding clamp, PCNA, carry out DNA synthesis during lagging strand replication, initiation of leading strand replication, and the major DNA damage repair and tolerance pathways. Pol δ holoenzymes are assembled at primer/template (P/T) junctions and initiate DNA synthesis in a stepwise process involving the major single strand DNA (ssDNA)-binding protein complex, RPA, the processivity sliding clamp loader, RFC, PCNA and pol δ. During this process, the interactions of RPA, RFC and pol δ with a P/T junction all significantly overlap. A burning issue that has yet to be resolved is how these overlapping interactions are accommodated during this process. To address this, we design and utilize novel, ensemble FRET assays that continuously monitor the interactions of RPA, RFC, PCNA and pol δ with DNA as pol δ holoenzymes are assembled and initiate DNA synthesis. Results from the present study reveal that RPA remains engaged with P/T junctions throughout this process and the RPA•DNA complexes dynamically re-organize to allow successive binding of RFC and pol δ. These results have broad implications as they highlight and distinguish the functional consequences of dynamic RPA•DNA interactions in RPA-dependent DNA metabolic processes.

Mechanistic insight into AP-endonuclease 1 cleavage of abasic sites at stalled replication fork mimics.

Many types of damage, including abasic sites, block replicative DNA polymerases causing replication fork uncoupling and generating ssDNA. AP-Endonuclease 1 (APE1) has been shown to cleave abasic sites in ssDNA. Importantly, APE1 cleavage of ssDNA at a replication fork has significant biological implications by generating double strand breaks that could collapse the replication fork. Despite this, the molecular basis and efficiency of APE1 processing abasic sites at replication forks remain elusive. Here, we investigate APE1 cleavage of abasic substrates that mimic APE1 interactions at stalled replication forks or gaps. We determine that APE1 has robust activity on these substrates, like dsDNA, and report rates for cleavage and product release. X-ray structures visualize the APE1 active site, highlighting an analogous mechanism is used to process ssDNA substrates as canonical APE1 activity on dsDNA. However, mutational analysis reveals R177 to be uniquely critical for the APE1 ssDNA cleavage mechanism. Additionally, we investigate the interplay between APE1 and Replication Protein A (RPA), the major ssDNA-binding protein at replication forks, revealing that APE1 can cleave an abasic site while RPA is still bound to the DNA. Together, this work provides molecular level insights into abasic ssDNA processing by APE1, including the presence of RPA.

Replication of [AT/TA]25 Microsatellite Sequences by Human DNA Polymerase δ Holoenzymes Is Dependent on dNTP and RPA Levels.

Fragile sites are unstable genomic regions that are prone to breakage during stressed DNA replication. Several common fragile sites (CFS) contain A+T-rich regions including perfect [AT/TA] microsatellite repeats that may collapse into hairpins when in single-stranded DNA (ssDNA) form and coincide with chromosomal hotspots for breakage and rearrangements. While many factors contribute to CFS instability, evidence exists for replication stalling within [AT/TA] microsatellite repeats. Currently, it is unknown how stress causes replication stalling within [AT/TA] microsatellite repeats. To investigate this, we utilized FRET to characterize the structures of [AT/TA]25 sequences and also reconstituted lagging strand replication to characterize the progression of pol δ holoenzymes through A+T-rich sequences. The results indicate that [AT/TA]25 sequences adopt hairpins that are unwound by the major ssDNA-binding complex, RPA, and the progression of pol δ holoenzymes through A+T-rich sequences saturated with RPA is dependent on the template sequence and dNTP concentration. Importantly, the effects of RPA on the replication of [AT/TA]25 sequences are dependent on dNTP concentration, whereas the effects of RPA on the replication of A+T-rich, nonstructure-forming sequences are independent of dNTP concentration. Collectively, these results reveal complexities in lagging strand replication and provide novel insights into how [AT/TA] microsatellite repeats contribute to genome instability.

Tracking of progressing human DNA polymerase δ holoenzymes reveals distributions of DNA lesion bypass activities.

During DNA replication, DNA lesions in lagging strand templates are initially encountered by DNA polymerase δ (pol δ) holoenzymes comprised of pol δ and the PCNA processivity sliding clamp. These encounters are thought to stall replication of an afflicted template before the lesion, activating DNA damage tolerance (DDT) pathways that replicate the lesion and adjacent DNA sequence, allowing pol δ to resume. However, qualitative studies observed that human pol δ can replicate various DNA lesions, albeit with unknown proficiencies, which raises issues regarding the role of DDT in replicating DNA lesions. To address these issues, we re-constituted human lagging strand replication to quantitatively characterize initial encounters of pol δ holoenzymes with DNA lesions. The results indicate pol δ holoenzymes support dNTP incorporation opposite and beyond multiple lesions and the extent of these activities depends on the lesion and pol δ proofreading. Furthermore, after encountering a given DNA lesion, subsequent dissociation of pol δ is distributed around the lesion and a portion does not dissociate. The distributions of these events are dependent on the lesion and pol δ proofreading. Collectively, these results reveal complexity and heterogeneity in the replication of lagging strand DNA lesions, significantly advancing our understanding of human DDT.

Structure of the human clamp loader reveals an autoinhibited conformation of a substrate-bound AAA+ switch.

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.

Structural basis of reiterative transcription from the pyrG and pyrBI promoters by bacterial RNA polymerase.

Reiterative transcription is a non-canonical form of RNA synthesis by RNA polymerase in which a ribonucleotide specified by a single base in the DNA template is repetitively added to the nascent RNA transcript. We previously determined the X-ray crystal structure of the bacterial RNA polymerase engaged in reiterative transcription from the pyrG promoter, which contains eight poly-G RNA bases synthesized using three C bases in the DNA as a template and extends RNA without displacement of the promoter recognition σ factor from the core enzyme. In this study, we determined a series of transcript initiation complex structures from the pyrG promoter using soak-trigger-freeze X-ray crystallography. We also performed biochemical assays to monitor template DNA translocation during RNA synthesis from the pyrG promoter and in vitro transcription assays to determine the length of poly-G RNA from the pyrG promoter variants. Our study revealed how RNA slips on template DNA and how RNA polymerase and template DNA determine length of reiterative RNA product. Lastly, we determined a structure of a transcript initiation complex at the pyrBI promoter and proposed an alternative mechanism of RNA slippage and extension requiring the σ dissociation from the core enzyme.

Rad6/Rad18 Competes with DNA Polymerases η and δ for PCNA Encircling DNA.

Translesion DNA synthesis (TLS) bypasses DNA lesions encountered during S-phase and is critical for cell survival after exposure to DNA-damaging agents. In humans, Rad6/Rad18 attaches single ubiquitin moieties (i.e., monoubiquitination) to proliferating cell nuclear antigen (PCNA) sliding clamps encircling primer/template (P/T) junctions that are stalled at DNA lesions. TLS occurs via PCNA monoubiquitination-independent and -dependent pathways, and both contribute to cell survival. The interaction of Rad6/Rad18 with PCNA is paramount to PCNA monoubiquitination and remains poorly defined. In particular, the location of the Rad6/Rad18 binding site on PCNA is unknown. Many PCNA-binding proteins, particularly DNA polymerases (pols), converge on PCNA encircling stalled P/T junctions in human cells, and all interact in a similar manner with the universal binding sites on PCNA. We reasoned the following: if Rad6/Rad18 utilizes the universal binding sites (or nearby sites), then PCNA monoubiquitination may be suppressed by pols involved in TLS. Results from quantitative studies reveal that (1) a Y-family pol (pol η) and a B-family pol (pol δ) critical to TLS each inhibit the transfer of ubiquitin from Rad6/Rad18 to PCNA and that (2) the observed inhibitions are dependent on the interaction of these pols with PCNA encircling DNA. These studies suggest that Rad6/Rad18 utilizes the universal PCNA-binding sites or nearby sites and, hence, competes for PCNA encircling DNA with pols η and δ and possibly other PCNA-binding proteins involved in TLS. These findings provide valuable insight into the nature of the interaction between Rad6/Rad18 and PCNA and have important implications for the division of human TLS pathways.

PCNA Monoubiquitination Is Regulated by Diffusion of Rad6/Rad18 Complexes along RPA Filaments.

Translesion DNA synthesis (TLS) enables DNA replication through damaging modifications to template DNA and requires monoubiquitination of the proliferating cell nuclear antigen (PCNA) sliding clamp by the Rad6/Rad18 complex. This posttranslational modification is critical to cell survival following exposure to DNA-damaging agents and is tightly regulated to restrict TLS to damaged DNA. Replication protein A (RPA), the major single-strand DNA (ssDNA) binding protein complex, forms filaments on ssDNA exposed at TLS sites and plays critical yet undefined roles in regulating PCNA monoubiquitination. Here, we utilize kinetic assays and single-molecule FRET microscopy to monitor PCNA monoubiquitination and Rad6/Rad18 complex dynamics on RPA filaments, respectively. Results reveal that a Rad6/Rad18 complex is recruited to an RPA filament via Rad18·RPA interactions and randomly translocates along the filament. These translocations promote productive interactions between the Rad6/Rad18 complex and the resident PCNA, significantly enhancing monoubiquitination. These results illuminate critical roles of RPA in the specificity and efficiency of PCNA monoubiquitination and represent, to the best of our knowledge, the first example of ATP-independent translocation of a protein complex along a protein filament.

Replication protein A dynamically regulates monoubiquitination of proliferating cell nuclear antigen.

DNA damage tolerance permits bypass of DNA lesions encountered during S-phase and may be carried out by translesion DNA synthesis (TLS). Human TLS requires selective monoubiquitination of proliferating cell nuclear antigen (PCNA) sliding clamps encircling damaged DNA. This posttranslational modification (PTM) is catalyzed by Rad6/Rad18. Recent studies revealed that replication protein A (RPA), the major ssDNA-binding protein, is involved in the regulation of PCNA monoubiquitination and interacts directly with Rad18 on chromatin and in the nucleoplasm. However, it is unclear how RPA regulates this critical PTM and what functional role(s) these interactions serve. Here, we developed an in vitro assay to quantitatively monitor PCNA monoubiquitination under in vivo scenarios. Results from extensive experiments revealed that RPA regulates Rad6/Rad18 activity in an ssDNA-dependent manner. We found that "DNA-free" RPA inhibits monoubiquitination of free PCNA by directly interacting with Rad18. This interaction is promoted under native conditions when there is an overabundance of free RPA in the nucleoplasm where Rad6/Rad18 and a significant fraction of PCNA reside. During DNA replication stress, RPA binds the ssDNA exposed downstream of stalled primer/template (P/T) junctions, releasing Rad6/Rad18. RPA restricted the resident PCNAs to the upstream duplex regions by physically blocking diffusion of PCNA along ssDNA, and this activity was required for efficient monoubiquitination of PCNA on DNA. Furthermore, upon binding ssDNA, RPA underwent a conformational change that increased its affinity for Rad18. Rad6/Rad18 complexed with ssDNA-bound RPA was active, and this interaction may selectively promote monoubiquitination of PCNA on long RPA-coated ssDNA.

Eukaryotic Translesion DNA Synthesis on the Leading and Lagging Strands: Unique Detours around the Same Obstacle.

During S-phase, minor DNA damage may be overcome by DNA damage tolerance (DDT) pathways that bypass such obstacles, postponing repair of the offending damage to complete the cell cycle and maintain cell survival. In translesion DNA synthesis (TLS), specialized DNA polymerases replicate the damaged DNA, allowing stringent DNA synthesis by a replicative polymerase to resume beyond the offending damage. Dysregulation of this DDT pathway in human cells leads to increased mutation rates that may contribute to the onset of cancer. Furthermore, TLS affords human cancer cells the ability to counteract chemotherapeutic agents that elicit cell death by damaging DNA in actively replicating cells. Currently, it is unclear how this critical pathway unfolds, in particular, where and when TLS occurs on each template strand. Given the semidiscontinuous nature of DNA replication, it is likely that TLS on the leading and lagging strand templates is unique for each strand. Since the discovery of DDT in the late 1960s, most studies on TLS in eukaryotes have focused on DNA lesions resulting from ultraviolet (UV) radiation exposure. In this review, we revisit these and other related studies to dissect the step-by-step intricacies of this complex process, provide our current understanding of TLS on leading and lagging strand templates, and propose testable hypotheses to gain further insights.

Stability of the human polymerase δ holoenzyme and its implications in lagging strand DNA synthesis.

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.

Replication Protein A Prohibits Diffusion of the PCNA Sliding Clamp along Single-Stranded DNA.

The replicative polymerases cannot accommodate distortions to the native DNA sequence such as modifications (lesions) to the native template bases from exposure to reactive metabolites and environmental mutagens. Consequently, DNA synthesis on an afflicted template abruptly stops upon encountering these lesions, but the replication fork progresses onward, exposing long stretches of the damaged template before eventually stalling. Such arrests may be overcome by translesion DNA synthesis (TLS) in which specialized TLS polymerases bind to the resident proliferating cell nuclear antigen (PCNA) and replicate the damaged DNA. Hence, a critical aspect of TLS is maintaining PCNA at or near a blocked primer/template (P/T) junction upon uncoupling of fork progression from DNA synthesis by the replicative polymerases. The single-stranded DNA (ssDNA) binding protein, replication protein A (RPA), coats the exposed template and might prohibit diffusion of PCNA along the single-stranded DNA adjacent to a blocked P/T junction. However, this idea had yet to be directly tested. We recently developed a unique Cy3-Cy5 Forster resonance energy transfer (FRET) pair that directly reports on the occupancy of DNA by PCNA. In this study, we utilized this FRET pair to directly and continuously monitor the retention of human PCNA at a blocked P/T junction. Results from extensive steady state and pre-steady state FRET assays indicate that RPA binds tightly to the ssDNA adjacent to a blocked P/T junction and restricts PCNA to the upstream duplex region by physically blocking diffusion of PCNA along ssDNA.

Monitoring the Retention of Human Proliferating Cell Nuclear Antigen at Primer/Template Junctions by Proteins That Bind Single-Stranded DNA.

In humans, proliferating cell nuclear antigen (PCNA) sliding clamps encircling DNA coordinate various aspects of DNA metabolism throughout the cell cycle. A critical aspect of this is restricting PCNA to the vicinity of its DNA target site. For example, PCNA must be maintained at or near primer/template (P/T) junctions during DNA synthesis. With a diverse array of cellular factors implicated, many of which interact with PCNA, DNA, or both, it is unknown how this critical feat is achieved. Furthermore, current biochemical assays that examine the retention of PCNA near P/T junctions are inefficient, discontinuous, and qualitative and significantly deviate from physiologically relevant conditions. To overcome these challenges and limitations, we recently developed a novel and convenient Förster resonance energy transfer (FRET) assay that directly and continuously monitors the retention of human PCNA at a P/T junction. Here we describe in detail the design, methodology, interpretation, and limitations of this quantitative FRET assay using the single-stranded DNA-binding protein, SSB, from Escherichia coli as an example. This powerful tool is broadly applicable to any single-stranded DNA-binding protein and may be utilized and/or expanded upon to dissect DNA metabolic pathways that are dependent upon PCNA.

Probing the DNA structural requirements for facilitated diffusion.

DNA glycosylases perform a genome-wide search to locate damaged nucleotides among a great excess of undamaged nucleotides. Many glycosylases are capable of facilitated diffusion, whereby multiple sites along the DNA are sampled during a single binding encounter. Electrostatic interactions between positively charged amino acids and the negatively charged phosphate backbone are crucial for facilitated diffusion, but the extent to which diffusing proteins rely on the double-helical structure DNA is not known. Kinetic assays were used to probe the DNA searching mechanism of human alkyladenine DNA glycosylase (AAG) and to test the extent to which diffusion requires B-form duplex DNA. Although AAG excises εA lesions from single-stranded DNA, it is not processive on single-stranded DNA because dissociation is faster than N-glycosidic bond cleavage. However, the AAG complex with single-stranded DNA is sufficiently stable to allow for DNA annealing when a complementary strand is added. This observation provides evidence of nonspecific association of AAG with single-stranded DNA. Single-strand gaps, bubbles, and bent structures do not impede the search by AAG. Instead, these flexible or bent structures lead to the capture of a nearby site of damage that is more efficient than that of a continuous B-form duplex. The ability of AAG to negotiate these helix discontinuities is inconsistent with a sliding mode of diffusion but can be readily explained by a hopping mode that involves microscopic dissociation and reassociation. These experiments provide evidence of relatively long-range hops that allow a searching protein to navigate around DNA binding proteins that would serve as obstacles to a sliding protein.

Isolating contributions from intersegmental transfer to DNA searching by alkyladenine DNA glycosylase.

Large genomes pose a challenge to DNA repair pathways because rare sites of damage must be efficiently located from among a vast excess of undamaged sites. Human alkyladenine DNA glycosylase (AAG) employs nonspecific DNA binding interactions and facilitated diffusion to conduct a highly redundant search of adjacent sites. This ensures that every site is searched, but could be a detriment if the protein is trapped in a local segment of DNA. Intersegmental transfer between DNA segments that are transiently in close proximity provides an elegant solution that balances global and local searching processes. It has been difficult to detect intersegmental transfer experimentally; therefore, we developed biochemical assays that allowed us to observe and measure the rates of intersegmental transfer by AAG. AAG has a flexible amino terminus that tunes its affinity for nonspecific DNA, but we find that it is not required for intersegmental transfer. As AAG has only a single DNA binding site, this argues against the bridging model for intersegmental transfer. The rates of intersegmental transfer are strongly dependent on the salt concentration, supporting a jumping mechanism that involves microscopic dissociation and capture by a proximal DNA site. As many DNA-binding proteins have only a single binding site, jumping may be a common mechanism for intersegmental transfer.

PARP2 promotes Break Induced Replication-mediated telomere fragility in response to replication stress.

PARP2 is a DNA-dependent ADP-ribosyl transferase (ARTs) enzyme with Poly(ADP-ribosyl)ation activity that is triggered by DNA breaks. It plays a role in the Base Excision Repair pathway, where it has overlapping functions with PARP1. However, additional roles for PARP2 have emerged in the response of cells to replication stress. In this study, we demonstrate that PARP2 promotes replication stress-induced telomere fragility and prevents telomere loss following chronic induction of oxidative DNA lesions and BLM helicase depletion. Telomere fragility results from the activity of the break-induced replication pathway (BIR). During this process, PARP2 promotes DNA end resection, strand invasion and BIR-dependent mitotic DNA synthesis by orchestrating POLD3 recruitment and activity. Our study has identified a role for PARP2 in the response to replication stress. This finding may lead to the development of therapeutic approaches that target DNA-dependent ART enzymes, particularly in cancer cells with high levels of replication stress.

Replication of [AT/TA]25 microsatellite sequences by human DNA polymerase δ holoenzymes is dependent on dNTP and RPA levels.

Difficult-to-Replicate Sequences (DiToRS) are natural impediments in the human genome that inhibit DNA replication under endogenous replication. Some of the most widely-studied DiToRS are A+T-rich, high "flexibility regions," including long stretches of perfect [AT/TA] microsatellite repeats that have the potential to collapse into hairpin structures when in single-stranded DNA (ssDNA) form and are sites of recurrent structural variation and double-stranded DNA (dsDNA) breaks. Currently, it is unclear how these flexibility regions impact DNA replication, greatly limiting our fundamental understanding of human genome stability. To investigate replication through flexibility regions, we utilized FRET to characterize the effects of the major ssDNA-binding complex, RPA, on the structure of perfect [AT/TA]25 microsatellite repeats and also re-constituted human lagging strand replication to quantitatively characterize initial encounters of pol δ holoenzymes with A+T-rich DNA template sequences. The results indicate that [AT/TA]25 sequences adopt hairpin structures that are unwound by RPA and pol δ holoenzymes support dNTP incorporation through the [AT/TA]25 sequences as well as an A+T-rich, non-structure forming sequence. Furthermore, the extent of dNTP incorporation is dependent on the sequence of the DNA template and the concentration of dNTPs. Importantly, the effects of RPA on the replication of [AT/TA]25 sequences are dependent on the concentration of dNTPs, whereas the effects of RPA on the replication of an A+T-rich, non-structure forming sequence are independent of dNTP concentration. Collectively, these results reveal complexities in lagging strand replication and provide novel insights into how flexibility regions contribute to genome instability.

Interplay of macromolecular interactions during assembly of human DNA polymerase δ holoenzymes and initiation of DNA synthesis.

In humans, DNA polymerase δ (Pol δ) holoenzymes, comprised of Pol δ and the processivity sliding clamp, proliferating cell nuclear antigen (PCNA), carry out DNA synthesis during lagging strand DNA replication, initiation of leading strand DNA replication, and the major DNA damage repair and tolerance pathways. Pol δ holoenzymes are assembled at primer/template (P/T) junctions and initiate DNA synthesis in a coordinated process involving the major single strand DNA-binding protein complex, replication protein A (RPA), the processivity sliding clamp loader, replication factor C (RFC), PCNA, and Pol δ. Each of these factors interact uniquely with a P/T junction and most directly engage one another. Currently, the interplay between these macromolecular interactions is largely unknown. In the present study, novel Förster Resonance Energy Transfer (FRET) assays reveal that dynamic interactions of RPA with a P/T junction during assembly of a Pol δ holoenzyme and initiation of DNA synthesis maintain RPA at a P/T junction and accommodate RFC, PCNA, and Pol δ, maximizing the efficiency of each process. Collectively, these studies significantly advance our understanding of human DNA replication and DNA repair.

Human alkyladenine DNA glycosylase employs a processive search for DNA damage.

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 strengths, 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 80 amino-terminal 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.