Human Bocavirus 1 NP1 acts as an ssDNA-binding protein to help AAV2 DNA replication and cooperates with RPA to regulate AAV2 capsid expression.

Adeno-associated virus (AAV) requires co-infection with helper virus for efficient replication. We previously reported that Human Bocavirus 1 (HBoV1) genes, including NP1, NS2, and BocaSR, were critical for AAV2 replication. Here, we first demonstrate the essential roles of the NP1 protein in AAV2 DNA replication and protein expression. We show that NP1 binds to single-strand DNA (ssDNA) at least 30 nucleotides (nt) in length in a sequence-independent manner. Furthermore, NP1 colocalized with the BrdU-labeled AAV2 DNA replication center, and the loss of the ssDNA-binding ability of NP1 by site-directed mutation completely abolished AAV2 DNA replication. We used affinity-tagged NP1 protein to identify host cellular proteins associated with NP1 in cells cotransfected with the HBoV1 helper genes and AAV2 duplex genome. Of the identified proteins, we demonstrate that NP1 directly binds to the DBD-F domain of the RPA70 subunit with a high affinity through the residues 101-121. By reconstituting the heterotrimer protein RPA in vitro using gel filtration, we demonstrate that NP1 physically associates with RPA to form a heterologous complex characterized by typical fast-on/fast-off kinetics. Following a dominant-negative strategy, we found that NP1-RPA complex mainly plays a role in expressing AAV2 capsid protein by enhancing the transcriptional activity of the p40 promoter. Our study revealed a novel mechanism by which HBoV1 NP1 protein supports AAV2 DNA replication and capsid protein expression through its ssDNA-binding ability and direct interaction with RPA, respectively.IMPORTANCERecombinant adeno-associated virus (rAAV) vectors have been extensively used in clinical gene therapy strategies. However, a limitation of these gene therapy strategies is the efficient production of the required vectors, as AAV alone is replication-deficient in the host cells. HBoV1 provides the simplest AAV2 helper genes consisting of NP1, NS2, and BocaSR. An important question regarding the helper function of HBoV1 is whether it provides any direct function that supports AAV2 DNA replication and protein expression. Also of interest is how HBoV1 interplays with potential host factors to constitute a permissive environment for AAV2 replication. Our studies revealed that the multifunctional protein NP1 plays important roles in AAV2 DNA replication via its sequence-independent ssDNA-binding ability and in regulating AAV2 capsid protein expression by physically interacting with host protein RPA. Our findings present theoretical guidance for the future application of the HBoV1 helper genes in the rAAV vector production.

Retrotransposons hijack alt-EJ for DNA replication and eccDNA biogenesis.

Retrotransposons are highly enriched in the animal genome1-3. The activation of retrotransposons can rewrite host DNA information and fundamentally impact host biology1-3. Although developmental activation of retrotransposons can offer benefits for the host, such as against virus infection, uncontrolled activation promotes disease or potentially drives ageing1-5. After activation, retrotransposons use their mRNA as templates to synthesize double-stranded DNA for making new insertions in the host genome1-3,6. Although the reverse transcriptase that they encode can synthesize the first-strand DNA1-3,6, how the second-strand DNA is generated remains largely unclear. Here we report that retrotransposons hijack the alternative end-joining (alt-EJ) DNA repair process of the host for a circularization step to synthesize their second-strand DNA. We used Nanopore sequencing to examine the fates of replicated retrotransposon DNA, and found that 10% of them achieve new insertions, whereas 90% exist as extrachromosomal circular DNA (eccDNA). Using eccDNA production as a readout, further genetic screens identified factors from alt-EJ as essential for retrotransposon replication. alt-EJ drives the second-strand synthesis of the long terminal repeat retrotransposon DNA through a circularization process and is therefore necessary for eccDNA production and new insertions. Together, our study reveals that alt-EJ is essential in driving the propagation of parasitic genomic retroelements. Our study uncovers a conserved function of this understudied DNA repair process, and provides a new perspective to understand-and potentially control-the retrotransposon life cycle.

Optimizing protein V untranslated region sequence in M13 phage for increased production of single-stranded DNA for origami.

DNA origami requires long scaffold DNA to be aligned with the guidance of short staple DNA strands. Scaffold DNA is produced in Escherichia coli as a form of the M13 bacteriophage by rolling circle amplification (RCA). This study shows that RCA can be reconfigured by reducing phage protein V (pV) expression, improving the production throughput of scaffold DNA by at least 5.66-fold. The change in pV expression was executed by modifying the untranslated region sequence and monitored using a reporter green fluorescence protein fused to pV. In a separate experiment, pV expression was controlled by an inducer. In both experiments, reduced pV expression was correlated with improved M13 bacteriophage production. High-cell-density cultivation was attempted for mass scaffold DNA production, and the produced scaffold DNA was successfully folded into a barrel shape without compromising structural quality. This result suggested that scaffold DNA production throughput can be significantly improved by reprogramming the RCA in E. coli.

High-throughput functional variant screens via in vivo production of single-stranded DNA.

Creating and characterizing individual genetic variants remains limited in scale, compared to the tremendous variation both existing in nature and envisioned by genome engineers. Here we introduce retron library recombineering (RLR), a methodology for high-throughput functional screens that surpasses the scale and specificity of CRISPR-Cas methods. We use the targeted reverse-transcription activity of retrons to produce single-stranded DNA (ssDNA) in vivo, incorporating edits at >90% efficiency and enabling multiplexed applications. RLR simultaneously introduces many genomic variants, producing pooled and barcoded variant libraries addressable by targeted deep sequencing. We use RLR for pooled phenotyping of synthesized antibiotic resistance alleles, demonstrating quantitative measurement of relative growth rates. We also perform RLR using the sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for causal variants, demonstrating that RLR is uniquely suited to utilize large pools of natural variation. Using ssDNA produced in vivo for pooled experiments presents avenues for exploring variation across the genome.

Structure and function of virion RNA polymerase of a crAss-like phage.

CrAss-like phages are a recently described expansive group of viruses that includes the most abundant virus in the human gut1-3. The genomes of all crAss-like phages encode a large virion-packaged protein2,4 that contains a DFDxD sequence motif, which forms the catalytic site in cellular multisubunit RNA polymerases (RNAPs)5. Here, using Cellulophaga baltica crAss-like phage phi14:2 as a model system, we show that this protein is a DNA-dependent RNAP that is translocated into the host cell along with the phage DNA and transcribes early phage genes. We determined the crystal structure of this 2,180-residue enzyme in a self-inhibited state, which probably occurs before virion packaging. This conformation is attained with the help of a cleft-blocking domain that interacts with the active site and occupies the cavity in which the RNA-DNA hybrid binds. Structurally, phi14:2 RNAP is most similar to eukaryotic RNAPs that are involved in RNA interference6,7, although most of the phi14:2 RNAP structure (nearly 1,600 residues) maps to a new region of the protein fold space. Considering this structural similarity, we propose that eukaryal RNA interference polymerases have their origins in phage, which parallels the emergence of the mitochondrial transcription apparatus8.

Synthesis of DNA Origami Scaffolds: Current and Emerging Strategies.

DNA origami nanocarriers have emerged as a promising tool for many biomedical applications, such as biosensing, targeted drug delivery, and cancer immunotherapy. These highly programmable nanoarchitectures are assembled into any shape or size with nanoscale precision by folding a single-stranded DNA scaffold with short complementary oligonucleotides. The standard scaffold strand used to fold DNA origami nanocarriers is usually the M13mp18 bacteriophage's circular single-stranded DNA genome with limited design flexibility in terms of the sequence and size of the final objects. However, with the recent progress in automated DNA origami design-allowing for increasing structural complexity-and the growing number of applications, the need for scalable methods to produce custom scaffolds has become crucial to overcome the limitations of traditional methods for scaffold production. Improved scaffold synthesis strategies will help to broaden the use of DNA origami for more biomedical applications. To this end, several techniques have been developed in recent years for the scalable synthesis of single stranded DNA scaffolds with custom lengths and sequences. This review focuses on these methods and the progress that has been made to address the challenges confronting custom scaffold production for large-scale DNA origami assembly.

Two components of DNA replication-dependent LexA cleavage.

Induction of the SOS response, a cellular system triggered by DNA damage in bacteria, depends on DNA replication for the generation of the SOS signal, ssDNA. RecA binds to ssDNA, forming filaments that stimulate proteolytic cleavage of the LexA transcriptional repressor, allowing expression of > 40 gene products involved in DNA repair and cell cycle regulation. Here, using a DNA replication system reconstituted in vitro in tandem with a LexA cleavage assay, we studied LexA cleavage during DNA replication of both undamaged and base-damaged templates. Only a ssDNA-RecA filament supported LexA cleavage. Surprisingly, replication of an undamaged template supported levels of LexA cleavage like that induced by a template carrying two site-specific cyclobutane pyrimidine dimers. We found that two processes generate ssDNA that could support LexA cleavage. 1) During unperturbed replication, single-stranded regions formed because of stochastic uncoupling of the leading-strand DNA polymerase from the replication fork DNA helicase, and 2) on the damaged template, nascent leading-strand gaps were generated by replisome lesion skipping. The two pathways differed in that RecF stimulated LexA cleavage during replication of the damaged template, but not normal replication. RecF appears to facilitate RecA filament formation on the leading-strand ssDNA gaps generated by replisome lesion skipping.

The iron-sulfur helicase DDX11 promotes the generation of single-stranded DNA for CHK1 activation.

The iron-sulfur (FeS) cluster helicase DDX11 is associated with a human disorder termed Warsaw Breakage Syndrome. Interestingly, one disease-associated mutation affects the highly conserved arginine-263 in the FeS cluster-binding motif. Here, we demonstrate that the FeS cluster in DDX11 is required for DNA binding, ATP hydrolysis, and DNA helicase activity, and that arginine-263 affects FeS cluster binding, most likely because of its positive charge. We further show that DDX11 interacts with the replication factors DNA polymerase delta and WDHD1. In vitro, DDX11 can remove DNA obstacles ahead of Pol δ in an ATPase- and FeS domain-dependent manner, and hence generate single-stranded DNA. Accordingly, depletion of DDX11 causes reduced levels of single-stranded DNA, a reduction of chromatin-bound replication protein A, and impaired CHK1 phosphorylation at serine-345. Taken together, we propose that DDX11 plays a role in dismantling secondary structures during DNA replication, thereby promoting CHK1 activation.

Dynamic Genome Editing Using In Vivo Synthesized Donor ssDNA in Escherichia coli.

As a key element of genome editing, donor DNA introduces the desired exogenous sequence while working with other crucial machinery such as CRISPR-Cas or recombinases. However, current methods for the delivery of donor DNA into cells are both inefficient and complicated. Here, we developed a new methodology that utilizes rolling circle replication and Cas9 mediated (RC-Cas-mediated) in vivo single strand DNA (ssDNA) synthesis. A single-gene rolling circle DNA replication system from Gram-negative bacteria was engineered to produce circular ssDNA from a Gram-positive parent plasmid at a designed sequence in Escherichia coli. Furthermore, it was demonstrated that the desired linear ssDNA fragment could be cut out using CRISPR-associated protein 9 (CRISPR-Cas9) nuclease and combined with lambda Red recombinase as donor for precise genome engineering. Various donor ssDNA fragments from hundreds to thousands of nucleotides in length were synthesized in E. coli cells, allowing successive genome editing in growing cells. We hope that this RC-Cas-mediated in vivo ssDNA on-site synthesis system will be widely adopted as a useful new tool for dynamic genome editing.

Bioproduction of pure, kilobase-scale single-stranded DNA.

Scalable production of kilobase single-stranded DNA (ssDNA) with sequence control has applications in therapeutics, gene synthesis and sequencing, scaffolded DNA origami, and archival DNA memory storage. Biological production of circular ssDNA (cssDNA) using M13 addresses these needs at low cost. However, one unmet goal is to minimize the essential protein coding regions of the exported DNA while maintaining its infectivity and production purity to produce sequences less than 3,000 nt in length, relevant to therapeutic and materials science applications. Toward this end, synthetic miniphage with inserts of custom sequence and size offers scalable, low-cost synthesis of cssDNA at milligram and higher scales. Here, we optimize growth conditions using an E. coli helper strain combined with a miniphage genome carrying only an f1 origin and a ß-lactamase-encoding (bla) antibiotic resistance gene, enabling isolation of pure cssDNA with a minimum sequence genomic length of 1,676 nt, without requiring additional purification from contaminating DNA. Low-cost scalability of isogenic, custom-length cssDNA is demonstrated for a sequence of 2,520 nt using a bioreactor, purified with low endotoxin levels (<5 E.U./ml). We apply these exonuclease-resistant cssDNAs to the self-assembly of wireframe DNA origami objects and to encode digital information on the miniphage genome for biological amplification.

Herpes simplex virus 1 ICP8 mutant lacking annealing activity is deficient for viral DNA replication.

Most DNA viruses that use recombination-dependent mechanisms to replicate their DNA encode a single-strand annealing protein (SSAP). The herpes simplex virus (HSV) single-strand DNA binding protein (SSB), ICP8, is the central player in all stages of DNA replication. ICP8 is a classical replicative SSB and interacts physically and/or functionally with the other viral replication proteins. Additionally, ICP8 can promote efficient annealing of complementary ssDNA and is thus considered to be a member of the SSAP family. The role of annealing during HSV infection has been difficult to assess in part, because it has not been possible to distinguish between the role of ICP8 as an SSAP from its role as a replicative SSB during viral replication. In this paper, we have characterized an ICP8 mutant, Q706A/F707A (QF), that lacks annealing activity but retains many other functions characteristic of replicative SSBs. Like WT ICP8, the QF mutant protein forms filaments in vitro, binds ssDNA cooperatively, and stimulates the activities of other replication proteins including the viral polymerase, helicase-primase complex, and the origin binding protein. Interestingly, the QF mutant does not complement an ICP8-null virus for viral growth, replication compartment formation, or DNA replication. Thus, we have been able to separate the activities of ICP8 as a replicative SSB from its annealing activity. Taken together, our data indicate that the annealing activity of ICP8 is essential for viral DNA replication in the context of infection and support the notion that HSV-1 uses recombination-dependent mechanisms during DNA replication.

Inhibition of checkpoint kinase 1 following gemcitabine-mediated S phase arrest results in CDC7- and CDK2-dependent replication catastrophe.

Combining DNA-damaging drugs with DNA checkpoint inhibitors is an emerging strategy to manage cancer. Checkpoint kinase 1 inhibitors (CHK1is) sensitize most cancer cell lines to DNA-damaging drugs and also elicit single-agent cytotoxicity in 15% of cell lines. Consequently, combination therapy may be effective in a broader patient population. Here, we characterized the molecular mechanism of sensitization to gemcitabine by the CHK1i MK8776. Brief gemcitabine incubation irreversibly inhibited ribonucleotide reductase, depleting dNTPs, resulting in durable S phase arrest. Addition of CHK1i 18 h after gemcitabine elicited cell division cycle 7 (CDC7)- and cyclin-dependent kinase 2 (CDK2)-dependent reactivation of the replicative helicase, but did not reinitiate DNA synthesis due to continued lack of dNTPs. Helicase reactivation generated extensive single-strand (ss)DNA that exceeded the protective capacity of the ssDNA-binding protein, replication protein A. The subsequent cleavage of unprotected ssDNA has been termed replication catastrophe. This mechanism did not occur with concurrent CHK1i plus gemcitabine treatment, providing support for delayed administration of CHK1i in patients. Alternative mechanisms of CHK1i-mediated sensitization to gemcitabine have been proposed, but their role was ruled out; these mechanisms include premature mitosis, inhibition of homologous recombination, and activation of double-strand break repair nuclease (MRE11). In contrast, single-agent activity of CHK1i was MRE11-dependent and was prevented by lower concentrations of a CDK2 inhibitor. Hence, both pathways require CDK2 but appear to depend on different CDK2 substrates. We conclude that a small-molecule inhibitor of CHK1 can elicit at least two distinct, context-dependent mechanisms of cytotoxicity in cancer cells.

Variable termination sites of DNA polymerases encountering a DNA-protein cross-link.

DNA-protein cross-links (DPCs) are important DNA lesions induced by endogenous crosslinking agents such as formaldehyde or acetaldehyde, as well as ionizing radiation, cancer chemotherapeutic drugs, and abortive action of some enzymes. Due to their very bulky nature, they are expected to interfere with DNA and RNA synthesis and DNA repair. DPCs are highly genotoxic and the ability of cells to deal with them is relevant for many chemotherapeutic interventions. However, interactions of DNA polymerases with DPCs have been poorly studied due to the lack of a convenient experimental model. We have used NaBH4-induced trapping of E. coli formamidopyrimidine-DNA glycosylase with DNA to construct model DNA polymerase substrates containing a DPC in single-stranded template, or in the template strand of double-stranded DNA, or in the non-template (displaced) strand of double-stranded DNA. Nine DNA polymerases belonging to families A, B, X, and Y were studied with respect to their behavior upon encountering a DPC: Klenow fragment of E. coli DNA polymerase I, Thermus aquaticus DNA polymerase I, Pyrococcus furiosus DNA polymerase, Sulfolobus solfataricus DNA polymerase IV, human DNA polymerases ß, κ and λ, and DNA polymerases from bacteriophages T4 and RB69. Although none were able to fully bypass DPCs in any context, Family B DNA polymerases (T4, RB69) and Family Y DNA polymerase IV were able to elongate the primer up to the site of the cross-link if a DPC was located in single-stranded template or in the displaced strand. In other cases, DNA synthesis stopped 4-5 nucleotides before the site of the cross-link in single-stranded template or in double-stranded DNA if the polymerases could displace the downstream strand. We suggest that termination of DNA polymerases on a DPC is mostly due to the unrelieved conformational strain experienced by the enzyme when pressing against the cross-linked protein molecule.

The mechanism of eukaryotic CMG helicase activation.

The initiation of eukaryotic DNA replication occurs in two discrete stages: first, the minichromosome maintenance (MCM) complex assembles as a head-to-head double hexamer that encircles duplex replication origin DNA during G1 phase; then, 'firing factors' convert each double hexamer into two active Cdc45-MCM-GINS helicases (CMG) during S phase. This second stage requires separation of the two origin DNA strands and remodelling of the double hexamer so that each MCM hexamer encircles a single DNA strand. Here we show that the MCM complex, which hydrolyses ATP during double-hexamer formation, remains stably bound to ADP in the double hexamer. Firing factors trigger ADP release, and subsequent ATP binding promotes stable CMG assembly. CMG assembly is accompanied by initial DNA untwisting and separation of the double hexamer into two discrete but inactive CMG helicases. Mcm10, together with ATP hydrolysis, then triggers further DNA untwisting and helicase activation. After activation, the two CMG helicases translocate in an 'N terminus-first' direction, and in doing so pass each other within the origin; this requires that each helicase is bound entirely to single-stranded DNA. Our experiments elucidate the mechanism of eukaryotic replicative helicase activation, which we propose provides a fail-safe mechanism for bidirectional replisome establishment.

DNA lesions proximity modulates damage tolerance pathways in Escherichia coli.

The genome of all organisms is constantly threatened by numerous agents that cause DNA damage. When the replication fork encounters an unrepaired DNA lesion, two DNA damage tolerance pathways are possible: error-prone translesion synthesis (TLS) that requires specialized DNA polymerases, and error-free damage avoidance that relies on homologous recombination (HR). The balance between these two mechanisms is essential since it defines the level of mutagenesis during lesion bypass, allowing genetic variability and adaptation to the environment, but also introduces the risk of generating genome instability. Here we report that the mere proximity of replication-blocking lesions that arise in Escherichia coli's genome during a genotoxic stress leads to a strong increase in the use of the error-prone TLS. We show that this increase is caused by the local inhibition of HR due to the overlapping of single-stranded DNA regions generated downstream of the lesions. This increase in TLS is independent of SOS activation, but its mutagenic effect is additive with the one of SOS. Hence, the combination of SOS induction and lesions proximity leads to a strong increase in TLS that becomes the main lesion tolerance pathway used by the cell during a genotoxic stress.

RNA-splicing factor SART3 regulates translesion DNA synthesis.

Translesion DNA synthesis (TLS) is one mode of DNA damage tolerance that uses specialized DNA polymerases to replicate damaged DNA. DNA polymerase η (Polη) is well known to facilitate TLS across ultraviolet (UV) irradiation and mutations in POLH are implicated in skin carcinogenesis. However, the basis for recruitment of Polη to stalled replication forks is not completely understood. In this study, we used an affinity purification approach to isolate a Polη-containing complex and have identified SART3, a pre-mRNA splicing factor, as a critical regulator to modulate the recruitment of Polη and its partner RAD18 after UV exposure. We show that SART3 interacts with Polη and RAD18 via its C-terminus. Moreover, SART3 can form homodimers to promote the Polη/RAD18 interaction and PCNA monoubiquitination, a key event in TLS. Depletion of SART3 also impairs UV-induced single-stranded DNA (ssDNA) generation and RPA focus formation, resulting in an impaired Polη recruitment and a higher mutation frequency and hypersensitivity after UV treatment. Notably, we found that several SART3 missense mutations in cancer samples lessen its stimulatory effect on PCNA monoubiquitination. Collectively, our findings establish SART3 as a novel Polη/RAD18 association regulator that protects cells from UV-induced DNA damage, which functions in a RNA binding-independent fashion.

Programmable autonomous synthesis of single-stranded DNA.

DNA performs diverse functional roles in biology, nanotechnology and biotechnology, but current methods for autonomously synthesizing arbitrary single-stranded DNA are limited. Here, we introduce the concept of primer exchange reaction (PER) cascades, which grow nascent single-stranded DNA with user-specified sequences following prescribed reaction pathways. PER synthesis happens in a programmable, autonomous, in situ and environmentally responsive fashion, providing a platform for engineering molecular circuits and devices with a wide range of sensing, monitoring, recording, signal-processing and actuation capabilities. We experimentally demonstrate a nanodevice that transduces the detection of a trigger RNA into the production of a DNAzyme that degrades an independent RNA substrate, a signal amplifier that conditionally synthesizes long fluorescent strands only in the presence of a particular RNA signal, molecular computing circuits that evaluate logic (AND, OR, NOT) combinations of RNA inputs, and a temporal molecular event recorder that records in the PER transcript the order in which distinct RNA inputs are sequentially detected.

Multi-copy single-stranded DNA in Escherichia coli.

Multi-copy single-stranded DNA (msDNA) is composed of covalently bound single-stranded DNA and RNA, and synthesized by retron-encoded reverse transcriptase. msDNA-synthesizing systems are thought to be a recent acquisition by Escherichia coli because, to date, only seven types of msDNA, which differ markedly in their primary nucleotide sequences, have been found in a small subset of E. coli strains. The wide use of E. coli in molecular research means that it is important to understand more about these stable, covalently bound, single-stranded DNA or RNA compounds. The present review provides insights into the molecular biosynthesis, distribution and function of E. coli msDNA to raise awareness about these special molecules.

Primer-Independent DNA Synthesis by a Family B DNA Polymerase from Self-Replicating Mobile Genetic Elements.

Family B DNA polymerases (PolBs) play a central role during replication of viral and cellular chromosomes. Here, we report the discovery of a third major group of PolBs, which we denote primer-independent PolB (piPolB), that might be a link between the previously known protein-primed and RNA/DNA-primed PolBs. PiPolBs are encoded by highly diverse mobile genetic elements, pipolins, integrated in the genomes of diverse bacteria and also present as circular plasmids in mitochondria. Biochemical characterization showed that piPolB displays efficient DNA polymerization activity that can use undamaged and damaged templates and is endowed with proofreading and strand displacement capacities. Remarkably, the protein is also capable of template-dependent de novo DNA synthesis, i.e., DNA-priming activity, thereby breaking the long-standing dogma that replicative DNA polymerases require a pre-existing primer for DNA synthesis. We suggest that piPolBs are involved in self-replication of pipolins and may also contribute to bacterial DNA damage tolerance.

The UL8 subunit of the helicase-primase complex of herpes simplex virus promotes DNA annealing and has a high affinity for replication forks.

During lytic infection, herpes simplex virus (HSV) DNA is replicated by a mechanism involving DNA recombination. For instance, replication of the HSV-1 genome produces X- and Y-branched structures, reminiscent of recombination intermediates. HSV-1's replication machinery includes a trimeric helicase-primase composed of helicase (UL5) and primase (UL52) subunits and a third subunit, UL8. UL8 has been reported to stimulate the helicase and primase activities of the complex in the presence of ICP8, an HSV-1 protein that functions as an annealase, a protein that binds complementary single-stranded DNA (ssDNA) and facilitates its annealing to duplex DNA. UL8 also influences the intracellular localization of the UL5/UL52 subunits, but UL8's catalytic activities are not known. In this study we used a combination of biochemical techniques and transmission electron microscopy. First, we report that UL8 alone forms protein filaments in solution. Moreover, we also found that UL8 binds to ssDNAs >50-nucletides long and promotes the annealing of complementary ssDNA to generate highly branched duplex DNA structures. Finally, UL8 has a very high affinity for replication fork structures containing a gap in the lagging strand as short as 15 nucleotides, suggesting that UL8 may aid in directing or loading the trimeric complex onto a replication fork. The properties of UL8 uncovered here suggest that UL8 may be involved in the generation of the X- and Y-branched structures that are the hallmarks of HSV replication.