Processing of Lagging-Strand Intermediates In Vitro by Herpes Simplex Virus Type 1 DNA Polymerase

The processing of lagging-strand intermediates has not been demonstrated in vitro for herpes simplex virus type 1 (HSV-1). Human flap endonuclease-1 (Fen-1) was examined for its ability to produce ligatable products with model lagging-strand intermediates in the presence of the wild-type or exonuclease-deficient (exo⁻) HSV-1 DNA polymerase (pol). Primer/templates were composed of a minicircle single-stranded DNA template annealed to primers that contained 5′ DNA flaps or 5′ annealed DNA or RNA sequences. Gapped DNA primer/templates were extended but not significantly strand displaced by the wild-type HSV-1 pol, although significant strand displacement was observed with exo⁻ HSV-1 pol. Nevertheless, the incubation of primer/templates containing 5′ flaps with either wild-type or exo⁻ HSV-1 pol and Fen-1 led to the efficient production of nicks that could be sealed with DNA ligase I. Both polymerases stimulated the nick translation activity of Fen-1 on DNA- or RNA-containing primer/templates, indicating that the activities were coordinated. Further evidence for Fen-1 involvement in HSV-1 DNA synthesis is suggested by the ability of a transiently expressed green fluorescent protein fusion with Fen-1 to accumulate in viral DNA replication compartments in infected cells and by the ability of endogenous Fen-1 to coimmunoprecipitate with an essential viral DNA replication protein in HSV-1-infected cells.Herpes simplex virus type 1 (HSV-1), the prototypic member of the family of Herpesviridae and that of the alphaherpesviridae subfamily, has served as the model for understanding the replication of herpesvirus genomes during lytic virus replication (29). The 152-kbp genome of herpes simplex virus type 1 (HSV-1) possesses approximately 85 genes, 7 of which have been shown to be necessary and sufficient for viral DNA replication within host cells (reviewed in references 5 and 38). These seven genes encode a DNA polymerase (pol) and its processivity factor (UL42), a heterotrimeric complex containing a DNA helicase (UL5), primase (UL52), and noncatalytic accessory protein (UL8), a single-stranded DNA binding protein (infected cell protein 8 [ICP-8]), and an origin binding protein with DNA helicase activity (UL9). There is strong evidence in support of the circularization of the linear virion DNA shortly after entry, and DNA replication then is thought to initiate at one or more of the three redundant origins of replication (29, 38). At least in the earliest stages of viral DNA replication, UL9 protein is required, presumably to bind to and unwind the DNA and to attract the other DNA replication proteins (29, 38). The electron microscopic examination of pulse-labeled replicating HSV-1 DNA indicates the presence of lariats, eye-forms, and D-forms (21), which is consistent with bidirectional theta-like replication from origins. To date, however, no biochemical assay has demonstrated origin-dependent DNA replication in vitro. However, in the absence of UL9, the other six HSV DNA replication proteins can support initiation and replication from a circular single-stranded DNA (ssDNA) template in an origin-independent fashion (15, 26), resembling the rolling-circle mode of replication thought to occur during the later stages of viral replication.Although nicks and small gaps have been observed in isolated replicating and virion DNA (38), the evidence for bidirectional duplex synthesis, the rapid rate of viral DNA replication, and the absence of long stretches of ssDNA in replicating and mature DNA isolated from HSV-1-infected cells suggest that leading- and lagging-strand synthesis are closely coordinated in vivo. Falkenberg et al. (15) used a minicircle DNA template with a strand bias and the six essential HSV-1 DNA replication proteins needed for rolling circle replication to demonstrate lagging-strand synthesis in vitro. However, replication from the parental strand template (leading-strand synthesis) was more efficient than synthesis from the complementary-strand template (lagging-strand synthesis). These results suggest the possibility that one or more host functions required for efficient lagging-strand synthesis or for its close coordination with leading-strand synthesis is missing in such in vitro systems.Although leading- and lagging-strand syntheses share many of the same requirements for bulk DNA synthesis, lagging-strand synthesis is a more complex process. Because the direction of polymerization of lagging-strand intermediates is opposite the direction of replication fork movement, lagging-strand synthesis requires that priming and extension occur many times to produce discontinuous segments called Okazaki fragments (reviewed in reference 25). Okazaki fragments need to be processed to remove the RNA primer, to fill in the area previously occupied by the RNA, and to seal the remaining nick between fragments, all of which must occur efficiently, accurately, and completely. Failure to do so would result in the accumulation of DNA breaks, multiple mutations, delayed DNA replication, and/or cell death (16, 61).In eukaryotes, what is currently known regarding the process of lagging-strand synthesis is based on genetic and biochemical studies with Saccharomyces cerevisiae and on in vitro reconstitution studies to define the mammalian enzymes required for simian virus 40 (SV40) T-antigen-dependent DNA replication (17, 37, 44, 57, 58). These studies have revealed that the extension of a newly synthesized Okazaki fragment DNA with pol δ causes the strand displacement of the preceding fragment to produce a 5′ flap (25). Results suggest that flap endonuclease 1 (Fen-1) is the activity responsible for the removal of the bulk of the 5′ flaps generated (1, 44, 48), although dna2 protein may facilitate the removal of longer flaps coated with the ssDNA binding protein complex (2, 44). In addition, the overexpression of exonuclease I can partially compensate for the loss of Fen-1 function in yeast (24, 51). For the proper processing of lagging-strand intermediates, the entire 5′ flap and all of the RNA primer need to be removed, and the gap must be filled to achieve a ligatable nick. DNA ligase I has been shown to be the enzyme involved in sealing Okazaki fragments in yeast and in humans (3, 31, 50, 56, 57). DNA ligase I requires a nick in which there is a 5′ phosphate on one end and a 3′ hydroxyl linked to a deoxyribose sugar entity on the other, and it works poorly in the presence of mismatches (54). The close coordination of Fen-1 and DNA ligase I activities for Okazaki fragment processing is facilitated by the interactions of these proteins with proliferating cell nuclear antigen (PCNA), the processivity factor for pol δ and ɛ (6, 30, 32, 46, 52, 53).HSV-1 does not appear to encode a protein with DNA ligase activity or one that can specifically cleave 5′ flaps, although it does encode a 5′-to-3′ exonuclease activity (UL12 [10, 20]) and a 3′-to-5′ exonuclease activity that is part of the HSV-1 pol catalytic subunit (27). As for most eukaryotes, RNA primers are essential for HSV-1 DNA synthesis, as demonstrated by the presence of oligoribonucleotides in replicating DNA in vivo (4), by the well-characterized ability of the UL52 protein in complex with the UL5 helicase activity to synthesize oligoribonucleotide primers on ssDNA in vitro (11, 13), and by the requirement of the conserved catalytic residues in the UL52 primase in vitro and in HSV-1-infected cells (14, 26). It is the strand displacement activity of pol δ that produces the 5′ flaps that are key to the removal of RNA primers during Okazaki fragment processing (6, 25). However, we previously demonstrated that wild-type HSV-1 DNA polymerase possesses poor strand displacement activity (62), in contrast to mammalian DNA pol δ (25). Thus, it is not apparent how RNA primers would be removed when encountered by HSV-1 pol during HSV-1 lagging-strand synthesis or how such intermediates would be processed.We wished to test the hypothesis that the nick translation activity of mammalian Fen-1 could function in collaboration with HSV-1 pol to facilitate the proper removal of RNA primers and/or short flaps to produce the ligatable products required for Okazaki fragment processing. In this report, we have examined the ability of wild-type and exonuclease-deficient (exo⁻) HSV-1 pol, which differ in their respective strand displacement activities, to extend model lagging-strand substrates in the presence or absence of mammalian Fen-1. Our results demonstrate that both wild-type and exo⁻ HSV-1 pol can cooperate with and enhance Fen-1 activity to achieve a ligatable nick in vitro. Moreover, colocalization and coimmunoprecipitation studies reveal a physical association of Fen-1 with HSV-1 DNA replication proteins, supporting a model for the involvement of Fen-1 in HSV-1 DNA replication.     

Strategy for Extracting DNA from Clay Soil and Detecting a Specific Target Sequence via Selective Enrichment and Real-Time (Quantitative) PCR Amplification

We present a simple strategy for isolating and accurately enumerating target DNA from high-clay-content soils: desorption with buffers, an optional magnetic capture hybridization step, and quantitation via real-time PCR. With the developed technique, μg quantities of DNA were extracted from mg samples of pure kaolinite and a field clay soil.Isolating and characterizing DNA sequences for use in molecular methods are integral to evaluating microbial community diversity in soil (6, 21, 22, 24, 37). Any isolation protocol should maximize nucleic acid isolation while minimizing copurification of enzymatic inhibitors. Although several methods that focus on extraction of total community DNA from environmental soil and water samples have been published (7, 21, 26, 34), the lack of a standard nucleic acid isolation protocol (32) reflects the difficulty in accomplishing these goals, most likely due to the complex nature of the soil environment.DNA extraction is especially difficult for soils containing clay (3, 5), given the tight binding of DNA strands to clay soil particles (7, 10, 20). Additionally, extracellular DNA binds to and is copurified with soil humic substances (10), which inhibit the activity of enzymes such as restriction endonucleases and DNA polymerase (6, 13, 23). Although clay-bound DNA can be PCR amplified in the absence of inhibitors (1), it is often the case that inhibitors are present in the soil environment, among them bilirubin, bile salts, urobilinogens, and polysaccharides (40). Of these inhibitors, humic substances have been found to be the most recalcitrant (36).A promising technique for isolating specific target sequences from soil particles and enzymatic inhibitors is the magnetic capture hybridization-PCR technique (MCH-PCR) presented by Jacobsen (19) and used to obtain high detection sensitivities (11, 38).We have found no evidence in the published literature of the use of MCH-PCR on soils that have high clay contents and here present a three-step strategy for isolating specific DNA sequences from the most difficult soil environment—clay that contains humic substances—and enumerating a specific target sequence from the crude extract.     

The Transition of Closely Opposed Lesions to Double-Strand Breaks during Long-Patch Base Excision Repair Is Prevented by the Coordinated Action of DNA Polymerase δ and Rad27/Fen1

DNA double-strand breaks can result from closely opposed breaks induced directly in complementary strands. Alternatively, double-strand breaks could be generated during repair of clustered damage, where the repair of closely opposed lesions has to be well coordinated. Using single and multiple mutants of Saccharomyces cerevisiae (budding yeast) that impede the interaction of DNA polymerase δ and the 5′-flap endonuclease Rad27/Fen1 with the PCNA sliding clamp, we show that the lack of coordination between these components during long-patch base excision repair of alkylation damage can result in many double-strand breaks within the chromosomes of nondividing haploid cells. This contrasts with the efficient repair of nonclustered methyl methanesulfonate-induced lesions, as measured by quantitative PCR and S1 nuclease cleavage of single-strand break sites. We conclude that closely opposed single-strand lesions are a unique threat to the genome and that repair of closely opposed strand damage requires greater spatial and temporal coordination between the participating proteins than does widely spaced damage in order to prevent the development of double-strand breaks.Endogenous metabolism or environmental factors such as oxidizing and alkylating agents can produce a wide variety of lesions in DNA. The genomes of mammalian cells experience from 10,000 to as many as 200,000 modifications per day (37, 44). Most lesions are repaired by a complex network of proteins that are part of an elaborate, multistep base excision repair (BER) system that generates single-strand break (SSB) intermediates. Importantly, defects in BER can lead to malignancies and can be associated with age-associated disease, especially neurodegeneration (60).BER is initiated by specific DNA N-glycosylases that remove damaged bases, yielding apurinic/apyrimidinic (AP) sites. Subsequent incision by AP endonucleases results in SSBs, and excision results in a single base gap as a repair intermediate (33, 53). SSBs are expected to be frequent in the genome due to the abundance of base damage as well as intermediates of repair, recombination, replication, and other DNA transactions (15, 16). Because they are generally repaired efficiently by BER and SSB repair enzymes (16, 57), SSBs per se may not be a major source of genome instability. However, if lesions are clustered, the formation of two closely spaced SSBs on opposing strands (or a single SSB and a modified nucleotide or AP site) might pose a special risk in terms of the potential to generate mutations or the possibility of conversion to double-strand breaks (DSBs), which are potent genotoxic lesions. Clustered lesions can arise within cells by chance association of random DNA lesions in a small region or the induction of multiple events in a narrow region, as found for ionizing radiation and various chemicals, such as those used in cancer treatments (47, 58, 59). While efficient BER is important for genome integrity, the repair must be well coordinated to avoid the generation of closely opposed SSB intermediates at closely spaced lesions that could result in the secondary generation of DSBs, especially since cells have limited DSB repair capacity (<50 DSBs/cell in the case of Saccharomyces cerevisiae) (48). While the impact of clustered lesions on repair of DNA has been examined in vitro by use of purified enzymes or cell extracts (13, 14, 27, 39, 56), there has been little opportunity to address specifically the repair of clustered lesions, except for those arising from UV damage (49).Whether formed directly from sugar damage or as BER intermediates, SSBs formed during the repair of base damage often possess 5′-deoxyribose phosphate (5′-dRP) ends that are not suitable for rejoining by DNA ligases (9, 15). In humans, removal and repair of 5′-dRP are accomplished by different combinations of proteins (3, 15) that result in short-patch repair, involving replacement of a single nucleotide (nt), or long-patch repair, involving 2 to 10 nt. The budding yeast Saccharomyces cerevisiae lacks a DNA polymerase β that provides AP lyase activity required for short-patch repair in mammalian cells. Instead, removal and repair of a 5′-dRP rely on the long-patch pathway, involving the successive actions of DNA polymerase δ (Pol δ) for strand displacement, the Rad27/Fen1 endonuclease to remove 5′ flaps, and DNA ligase (Cdc9) to rejoin the resulting nicks (9). The sliding clamp protein PCNA, which interacts with all three players, has been proposed to play a central role in coordinating these processes (18, 19, 34). The coupling between the strand displacement reaction by Pol δ and the flap cutting reaction by Fen1 is highly efficient, with over 90% of the products released by Fen1 being mononucleotides (17).Although the coordination of Pol δ, PCNA, and Rad27/Fen1 provides efficient processing of individual lesions in DNA, closely opposed SSBs that arise during repair of base damage could manifest as DSBs, either directly or as a result of SSB processing. A DNA damaging agent that has been used frequently to characterize long- and short-patch BER is methyl methanesulfonate (MMS). Recently, we described the detection of closely opposed MMS-induced lesions in yeast (42). Since the closely opposed lesions might represent a special challenge to BER, we considered the possibility that they might specifically impact long-patch repair through Pol δ and/or coordination of events with Rad27/Fen1. Pol δ of S. cerevisiae is a heterotrimeric enzyme consisting of Pol3, Pol31, and Pol32 (23). The nonessential Pol32 subunit is involved in translesion DNA synthesis (TLS) (24, 30) and also break-induced replication (41). However, its role in other types of DNA repair remains unclear. Using our in vivo assay for specifically detecting closely spaced methylated DNA lesions (42) and SSBs, we examined the role of Pol32 as well as the cooperation between Pol δ, Rad27/Fen1, and PCNA in the repair of clustered DNA lesions induced by MMS in G₁ stationary-phase haploid yeast. We found that Pol32 plays an important role in ensuring that clustered lesions are efficiently repaired and do not transition to DSBs.     

Rapid Identification of Genes Encoding DNA Polymerases by Function-Based Screening of Metagenomic Libraries Derived from Glacial Ice

Small-insert and large-insert metagenomic libraries were constructed from glacial ice of the Northern Schneeferner, which is located on the Zugspitzplatt in Germany. Subsequently, these libraries were screened for the presence of DNA polymerase-encoding genes by complementation of an Escherichia coli polA mutant. Nine novel genes encoding complete DNA polymerase I proteins or domains typical of these proteins were recovered.DNA polymerases are essential for DNA replication and DNA repair. Based on sequence similarities and phylogenetic relationships, DNA polymerases are grouped into six different families (A, B, C, D, X, and Y) (17). In this study, we used a DNA polymerase I (polA) mutant of Escherichia coli as a host for the screening of metagenomic libraries. PolA belongs to family A and contains three different domains: a 5′-3′ exonuclease domain at the N terminus, a central proofreading 3′-5′ exonuclease domain, and a polymerase domain at the C terminus of the enzyme (11). These polymerases are employed as tools in molecular biology, including probe labeling, DNA sequencing, and mutagenic PCR (13). To improve their suitability for such applications, various family A DNA polymerases have been modified; e.g., the Klenow fragment of E. coli DNA polymerase I has been redesigned by the removal of the 5′-3′ exonuclease domain (12). Nevertheless, expanding the known DNA polymerase sequence space and discovery of polymerases with novel properties are required for the development of novel or improved molecular methods and tools (13, 20).Metagenomics based on direct isolation of DNA from environmental samples, generation of metagenomic libraries from the isolated DNA, and function-based screening of the constructed libraries has led to identification and characterization of a variety of novel biocatalysts, such as lipases, amylases, amidases, nitrilases, and oxidoreductases (for reviews, see references 6, 7, and 10). In particular, the use of host strains or mutants of host strains that require heterologous complementation for growth under selective conditions has proven to be an efficient strategy to screen complex metagenomic libraries. This approach has been applied to, e.g., the isolation of genes encoding Na⁺/H⁺ antiporters (14), antibiotic resistance (18), or enzymes involved in poly-3-hydroxybutyrate metabolism (21).In this study, we employed the last-named strategy to recover functional genes encoding DNA polymerases. To our knowledge, this is the first report of identification of polymerases or other DNA-modifying enzymes by function-driven screening of metagenomes. For this purpose, we constructed small-insert and large-insert metagenomic libraries from DNA isolated from glacial ice. The employment of glacial ice samples for metagenomic library construction has not been reported by other researchers. The screening for the targeted genes was based on complementation of a cold-sensitive lethal mutation in the polA gene of E. coli (16).