Lagging Strand DNA Replication Research Articles

BLM helicase unwinds lagging strand substrates to assemble the ALT telomere damage response

The Bloom syndrome (BLM) helicase is critical for alternative lengthening of telomeres (ALT), a homology-directed repair (HDR)-mediated telomere maintenance mechanism that is prevalent in cancers of mesenchymal origin. The DNA substrates that BLM engages to direct telomere recombination during ALT remain unknown. Here, we determine that BLM helicase acts on lagging strand telomere intermediates that occur specifically in ALT-positive cells to assemble a replication-associated DNA damage response. Loss of ATRX was permissive for BLM localization to ALT telomeres in S and G2, commensurate with the appearance of telomere C-strand-specific single-stranded DNA (ssDNA). DNA2 nuclease deficiency increased 5'-flap formation in a BLM-dependent manner, while telomere C-strand, but not G-strand, nicks promoted ALT. These findings define the seminal events in the ALT DNA damage response, linking aberrant telomeric lagging strand DNA replication with a BLM-directed HDR mechanism that sustains telomere length in a subset of human cancers.

Abstract 418: BLM helicase unwinds lagging strand substrates to assemble the ALT telomere damage response

Abstract The Bloom Syndrome helicase (BLM) is critical for Alternative Lengthening of Telomeres (ALT), a homology directed repair mediated telomere maintenance mechanism that is prevalent in cancers of mesenchymal origin. The DNA substrates that BLM engages to direct telomere recombination during ALT remain unknown. Here, we determine that BLM helicase acts on lagging strand telomere intermediates that occur specifically in ALT positive cells to assemble a replication stress associated DNA damage response. Loss of ATRX was permissive for BLM localization to ALT telomeres in S and G2 commensurate with the appearance of telomere C-strand specific single-stranded DNA. Moreover, DNA2 nuclease deficiency increased 5’-lagging strand flap formation and ALT in a BLM dependent manner, and BLM promoted ALT in response to telomere C-strand, but not G-strand, single-stranded nicks. These findings define the seminal events in the ALT DNA damage response, linking aberrant lagging strand DNA replication with a BLM directed HDR mechanism that sustains telomere length in a subset of human cancers. Citation Format: Aravind M. Krishnan, Haoyang Jiang, Tianpeng Zhang, Hardeep Kaur, Tao Shi, Youngho Kwon, Patrick Sung, Roger A. Greenberg. BLM helicase unwinds lagging strand substrates to assemble the ALT telomere damage response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 418.

CST-polymerase α-primase solves a second telomere end-replication problem.

Telomerase adds G-rich telomeric repeats to the 3' ends of telomeres1, counteracting telomere shortening caused by loss of telomeric 3' overhangs during leading-strand DNA synthesis ('the end-replication problem'2). Here we report a second end-replication problem that originates from the incomplete duplication of the C-rich telomeric repeat strand (C-strand) by lagging-strand DNA synthesis. This problem is resolved by fill-in synthesis mediated by polymeraseα-primase bound to Ctc1-Stn1-Ten1 (CST-Polα-primase). In vitro, priming for lagging-strand DNA replication does not occur on the 3' overhang and lagging-strand synthesis stops in a zone of approximately 150 nucleotides (nt) more than 26 nt from the end of the template. Consistent with the in vitro data, lagging-end telomeres of cells lacking CST-Polα-primase lost 50-60 nt of telomeric CCCTAA repeats per population doubling. The C-strands of leading-end telomeres shortened by around 100 nt per population doubling, reflecting the generation of 3' overhangs through resection. The measured overall C-strand shortening in the absence of CST-Polα-primase fill-in is consistent with the combined effects of incomplete lagging-strand synthesis and 5' resection at the leading ends. We conclude that canonical DNA replication creates two telomere end-replication problems that require telomerase to maintain the G-rich strand and CST-Polα-primase to maintain the C-strand.

Abstract B019: BLM helicase unwinds lagging strand substrates to assemble the ALT telomere damage response

Abstract Alternative Lengthening of Telomeres (ALT) is a homology-directed repair (HDR) mediated telomere maintenance mechanism that is prevalent in cancers of mesenchymal origin. ALT cells exhibit more endogenous replication stress and DNA damage at the telomere compared to ALT-negative cells. The Bloom Syndrome helicase (BLM) is critical for ALT and promotes replication stress and DNA damage specifically at telomeres in ALT cells. Here, we determine that BLM helicase acts on lagging strand telomere intermediates that occur specifically in ALT-positive cells to assemble a replication stress-associated DNA damage response. Loss of ATRX was permissive for BLM localization to ALT telomeres in S and G2 commensurate with the appearance of telomere C-strand specific single-stranded DNA. Moreover, DNA2 nuclease deficiency increased 5’-flap formation in a BLM-dependent manner, and BLM promoted ALT in response to telomere C-strand, but not G-strand, nicks. These findings define the seminal events in the ALT DNA damage response, linking aberrant lagging strand DNA replication with a BLM-directed HDR mechanism that sustains telomere length in a subset of human cancers (Jiang et al. submitted). These findings will be discussed and how BLM activity is a key determinant of the specific vulnerability of ALT cells to FANCM inhibition, which is currently being developed to target ALT-dependent cancers. Citation Format: Haoyang Jiang, Tianpeng Zhang, Hardeep Kaur, Tao Shi, Aravind Krishnan, Youngho Kwon, Patrick Sung, Roger Greenberg. BLM helicase unwinds lagging strand substrates to assemble the ALT telomere damage response [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B019.

DNA Polymerase Delta Exhibits Altered Catalytic Properties on Lysine Acetylation.

DNA polymerase delta is the primary polymerase that is involved in undamaged nuclear lagging strand DNA replication. Our mass-spectroscopic analysis has revealed that the human DNA polymerase δ is acetylated on subunits p125, p68, and p12. Using substrates that simulate Okazaki fragment intermediates, we studied alterations in the catalytic properties of acetylated polymerase and compared it to the unmodified form. The current data show that the acetylated form of human pol δ displays a higher polymerization activity compared to the unmodified form of the enzyme. Additionally, acetylation enhances the ability of the polymerase to resolve complex structures such as G-quadruplexes and other secondary structures that might be present on the template strand. More importantly, the ability of pol δ to displace a downstream DNA fragment is enhanced upon acetylation. Our current results suggest that acetylation has a profound effect on the activity of pol δ and supports the hypothesis that acetylation may promote higher-fidelity DNA replication.

Fast and efficient DNA replication with purified human proteins.

Chromosome replication is performed by a complex and intricate ensemble of proteins termed the replisome, where the DNA polymerases Polδ and Polε, DNA polymerase α-primase (Polα) and accessory proteins including AND-1, CLASPIN and TIMELESS-TIPIN (respectively known as Ctf4, Mrc1 and Tof1-Csm3 in Saccharomyces cerevisiae) are organized around the CDC45-MCM-GINS (CMG) replicative helicase1-7. Because a functional human replisome has not been reconstituted from purified proteins, how these factors contribute to human DNA replication and whether additional proteins are required for optimal DNA synthesis are poorly understood. Here we report the biochemical reconstitution of human replisomes that perform fast and efficient DNA replication using 11 purified human replication factors made from 43 polypeptides. Polε, but not Polδ, is crucial for optimal leading-strand synthesis. Unexpectedly, Polε-mediated leading-strand replication is highly dependent on the sliding-clamp processivity factor PCNA and the alternative clamp loader complex CTF18-RFC. We show how CLASPIN and TIMELESS-TIPIN contribute to replisome progression and demonstrate that, in contrast to the budding yeast replisome8, AND-1 directly augments leading-strand replication. Moreover, although AND-1 binds to Polα9,10, the interaction is dispensable for lagging-strand replication, indicating that Polα is functionally recruited via an AND-1-independent mechanism for priming in the human replisome. Collectively, our work reveals how the human replisome achieves fast and efficient leading-strand and lagging-strand DNA replication, and provides a powerful system for future studies of the human replisome and its interactions with other DNA metabolic processes.

SV40 T antigen interactions with ssDNA and replication protein A: a regulatory role of T antigen monomers in lagging strand DNA replication.

DNA replication is a central process in all living organisms. Polyomavirus DNA replication serves as a model system for eukaryotic DNA replication and has considerably contributed to our understanding of basic replication mechanisms. However, the details of the involved processes are still unclear, in particular regarding lagging strand synthesis. To delineate the complex mechanism of coordination of various cellular proteins binding simultaneously or consecutively to DNA to initiate replication, we investigated single-stranded DNA (ssDNA) interactions by the SV40 large T antigen (Tag). Using single molecule imaging by atomic force microscopy (AFM) combined with biochemical and spectroscopic analyses we reveal independent activity of monomeric and oligomeric Tag in high affinity binding to ssDNA. Depending on ssDNA length, we obtain dissociation constants for Tag-ssDNA interactions (KD values of 10–30 nM) that are in the same order of magnitude as ssDNA binding by human replication protein A (RPA). Furthermore, we observe the formation of RPA-Tag-ssDNA complexes containing hexameric as well as monomeric Tag forms. Importantly, our data clearly show stimulation of primase function in lagging strand Okazaki fragment synthesis by monomeric Tag whereas hexameric Tag inhibits the reaction, redefining DNA replication initiation on the lagging strand.

Genome-wide de novo L1 Retrotransposition Connects Endonuclease Activity with Replication

L1 retrotransposon-derived sequences comprise approximately 17% of the human genome. Darwinian selective pressures alter L1 genomic distributions during evolution, confounding the ability to determine initial L1 integration preferences. Here, we generated high-confidence datasets of greater than 88,000 engineered L1 insertions in human cell lines that act as proxies for cells that accommodate retrotransposition invivo. Comparing these insertions to a null model, inwhich L1 endonuclease activity is the sole determinant dictating L1 integration preferences, demonstrated that L1 insertions are not significantly enriched in genes, transcribed regions, or open chromatin. By comparison, we provide compelling evidence that the L1 endonuclease disproportionately cleaves predominant lagging strand DNA replication templates, while lagging strand 3'-hydroxyl groups may prime endonuclease-independent L1 retrotransposition in a Fanconi anemia cell line. Thus, acquisition of an endonuclease domain, in conjunction with the ability to integrate into replicating DNA, allowed L1 to become an autonomous, interspersed retrotransposon.

High-accuracy lagging-strand DNA replication mediated by DNA polymerase dissociation

The fidelity of DNA replication is a critical factor in the rate at which cells incur mutations. Due to the antiparallel orientation of the two chromosomal DNA strands, one strand (leading strand) is replicated in a mostly processive manner, while the other (lagging strand) is synthesized in short sections called Okazaki fragments. A fundamental question that remains to be answered is whether the two strands are copied with the same intrinsic fidelity. In most experimental systems, this question is difficult to answer, as the replication complex contains a different DNA polymerase for each strand, such as, for example, DNA polymerases δ and ε in eukaryotes. Here we have investigated this question in the bacterium Escherichia coli, in which the replicase (DNA polymerase III holoenzyme) contains two copies of the same polymerase (Pol III, the dnaE gene product), and hence the two strands are copied by the same polymerase. Our in vivo mutagenesis data indicate that the two DNA strands are not copied with the same accuracy, and that, remarkably, the lagging strand has the highest fidelity. We postulate that this effect results from the greater dissociative character of the lagging-strand polymerase, which provides additional options for error removal. Our conclusion is strongly supported by results with dnaE antimutator polymerases characterized by increased dissociation rates.

Plant organellar DNA primase-helicase synthesizes RNA primers for organellar DNA polymerases using a unique recognition sequence.

DNA primases recognize single-stranded DNA (ssDNA) sequences to synthesize RNA primers during lagging-strand replication. Arabidopsis thaliana encodes an ortholog of the DNA primase-helicase from bacteriophage T7, dubbed AtTwinkle, that localizes in chloroplasts and mitochondria. Herein, we report that AtTwinkle synthesizes RNA primers from a 5′-(G/C)GGA-3′ template sequence. Within this sequence, the underlined nucleotides are cryptic, meaning that they are essential for template recognition but are not instructional during RNA synthesis. Thus, in contrast to all primases characterized to date, the sequence recognized by AtTwinkle requires two nucleotides (5′-GA-3′) as a cryptic element. The divergent zinc finger binding domain (ZBD) of the primase module of AtTwinkle may be responsible for template sequence recognition. During oligoribonucleotide synthesis, AtTwinkle shows a strong preference for rCTP as its initial ribonucleotide and a moderate preference for rGMP or rCMP incorporation during elongation. RNA products synthetized by AtTwinkle are efficiently used as primers for plant organellar DNA polymerases. In sum, our data strongly suggest that AtTwinkle primes organellar DNA polymerases during lagging strand synthesis in plant mitochondria and chloroplast following a primase-mediated mechanism. This mechanism contrasts to lagging-strand DNA replication in metazoan mitochondria, in which transcripts synthesized by mitochondrial RNA polymerase prime mitochondrial DNA polymerase γ.

Arranging eukaryotic nuclear DNA polymerases for replication: Specific interactions with accessory proteins arrange Pols α, δ, and ϵ in the replisome for leading-strand and lagging-strand DNA replication.

Biochemical and cryo-electron microscopy studies have just been published revealing interactions among proteins of the yeast replisome that are important for highly coordinated synthesis of the two DNA strands of the nuclear genome. These studies reveal key interactions important for arranging DNA polymerases α, δ, and ϵ for leading and lagging strand replication. The CMG (Mcm2-7, Cdc45, GINS) helicase is central to this interaction network. These are but the latest examples of elegant studies performed in the recent past that lead to a much better understanding of how the eukaryotic replication fork achieves efficient DNA replication that is accurate enough to prevent diseases yet allows evolution.

The GAN Exonuclease or the Flap Endonuclease Fen1 and RNase HII Are Necessary for Viability of Thermococcus kodakarensis.

Many aspects of and factors required for DNA replication are conserved across all three domains of life, but there are some significant differences surrounding lagging-strand synthesis. In Archaea, a 5'-to-3' exonuclease, related to both bacterial RecJ and eukaryotic Cdc45, that associates with the replisome specifically through interactions with GINS was identified and designated GAN (for GINS-associated nuclease). Despite the presence of a well-characterized flap endonuclease (Fen1), it was hypothesized that GAN might participate in primer removal during Okazaki fragment maturation, and as a Cdc45 homologue, GAN might also be a structural component of an archaeal CMG (Cdc45, MCM, and GINS) replication complex. We demonstrate here that, individually, either Fen1 or GAN can be deleted, with no discernible effects on viability and growth. However, deletion of both Fen1 and GAN was not possible, consistent with both enzymes catalyzing the same step in primer removal from Okazaki fragments in vivo RNase HII has also been proposed to participate in primer processing during Okazaki fragment maturation. Strains with both Fen1 and RNase HII deleted grew well. GAN activity is therefore sufficient for viability in the absence of both RNase HII and Fen1, but it was not possible to construct a strain with both RNase HII and GAN deleted. Fen1 alone is therefore insufficient for viability in the absence of both RNase HII and GAN. The ability to delete GAN demonstrates that GAN is not required for the activation or stability of the archaeal MCM replicative helicase.IMPORTANCE The mechanisms used to remove primer sequences from Okazaki fragments during lagging-strand DNA replication differ in the biological domains. Bacteria use the exonuclease activity of DNA polymerase I, whereas eukaryotes and archaea encode a flap endonuclease (Fen1) that cleaves displaced primer sequences. RNase HII and the GINS-associated exonuclease GAN have also been hypothesized to assist in primer removal in Archaea Here we demonstrate that in Thermococcus kodakarensis, either Fen1 or GAN activity is sufficient for viability. Furthermore, GAN can support growth in the absence of both Fen1 and RNase HII, but Fen1 and RNase HII are required for viability in the absence of GAN.

Mechanism of Lagging-Strand DNA Replication in Eukaryotes.

This chapter focuses on the enzymes and mechanisms involved in lagging-strand DNA replication in eukaryotic cells. Recent structural and biochemical progress with DNA polymerase α-primase (Pol α) provides insights how each of the millions of Okazaki fragments in a mammalian cell is primed by the primase subunit and further extended by its polymerase subunit. Rapid kinetic studies of Okazaki fragment elongation by Pol δ illuminate events when the polymerase encounters the double-stranded RNA-DNA block of the preceding Okazaki fragment. This block acts as a progressive molecular break that provides both time and opportunity for the flap endonuclease 1 (FEN1) to access the nascent flap and cut it. The iterative action of Pol δ and FEN1 is coordinated by the replication clamp PCNA and produces a regulated degradation of the RNA primer, thereby preventing the formation of long-strand displacement flaps. Occasional long flaps are further processed by backup nucleases including Dna2.

Suppression of mitochondrial transcription initiation complexes changes the balance of replication intermediates of mitochondrial DNA and reduces 7S DNA in cultured human cells.

Analysis of replicating mammalian mitochondrial DNA (mtDNA) suggested that initiation of the replication occurs not only at the specific position, Ori-H but also across a broad zone in mtDNA. We investigated relationship of mitochondrial transcription initiation which takes place upstream of Ori-H and mtDNA replication initiation through analysing the effect of knockdown of mitochondrial transcription factor B2, TFB2M and mitochondrial RNA polymerase, POLRMT, components of the transcription initiation complexes in cultured human cells. Under the conditions where suppression of the transcription initiation complexes was achieved by simultaneous depletion of TFB2M and POLRMT, decrease of replication intermediates of mtDNA RITOLS replication mode accompanied reduction in mtDNA copy number. On the other hand, replication intermediates of coupled leading and lagging strand DNA replication, another proposed replication mode, appeared to be less affected. The findings support the view that the former mode involves transcription from the light strand promoter (LSP), and suggest that initiation of the latter mode is independent from the transcription and has distinct regulation. Further, knockdown of TFB2M alone caused significant decrease of 7S DNA, which implies that transcription initiation complexes formed at the LSP engage 7S DNA synthesis more frequently than the initiation of productive replication and transcription.

Genetic Networks Required to Coordinate Chromosome Replication by DNA Polymerases α, δ, and ε in Saccharomyces cerevisiae.

Three major DNA polymerases replicate the linear eukaryotic chromosomes. DNA polymerase α-primase (Pol α) and DNA polymerase δ (Pol δ) replicate the lagging-strand and Pol α and DNA polymerase ε (Pol ε) the leading-strand. To identify factors affecting coordination of DNA replication, we have performed genome-wide quantitative fitness analyses of budding yeast cells containing defective polymerases. We combined temperature-sensitive mutations affecting the three replicative polymerases, Pol α, Pol δ, and Pol ε with genome-wide collections of null and reduced function mutations. We identify large numbers of genetic interactions that inform about the roles that specific genes play to help Pol α, Pol δ, and Pol ε function. Surprisingly, the overlap between the genetic networks affecting the three DNA polymerases does not represent the majority of the genetic interactions identified. Instead our data support a model for division of labor between the different DNA polymerases during DNA replication. For example, our genetic interaction data are consistent with biochemical data showing that Pol ε is more important to the Pre-Loading complex than either Pol α or Pol δ. We also observed distinct patterns of genetic interactions between leading- and lagging-strand DNA polymerases, with particular genes being important for coupling proliferating cell nuclear antigen loading/unloading (Ctf18, Elg1) with nucleosome assembly (chromatin assembly factor 1, histone regulatory HIR complex). Overall our data reveal specialized genetic networks that affect different aspects of leading- and lagging-strand DNA replication. To help others to engage with these data we have generated two novel, interactive visualization tools, DIXY and Profilyzer.

Flap Endonuclease 1 Limits Telomere Fragility on the Leading Strand

The existence of redundant replication and repair systems that ensure genome stability underscores the importance of faithful DNA replication. Nowhere is this complexity more evident than in challenging DNA templates, including highly repetitive or transcribed sequences. Here, we demonstrate that flap endonuclease 1 (FEN1), a canonical lagging strand DNA replication protein, is required for normal, complete leading strand replication at telomeres. We find that the loss of FEN1 nuclease activity, but not DNA repair activities, results in leading strand-specific telomere fragility. Furthermore, we show that FEN1 depletion-induced telomere fragility is increased by RNA polymerase II inhibition and is rescued by ectopic RNase H1 expression. These data suggest that FEN1 limits leading strand-specific telomere fragility by processing RNA:DNA hybrid/flap intermediates that arise from co-directional collisions occurring between the replisome and RNA polymerase. Our data reveal the first molecular mechanism for leading strand-specific telomere fragility and the first known role for FEN1 in leading strand DNA replication. Because FEN1 mutations have been identified in human cancers, our findings raise the possibility that unresolved RNA:DNA hybrid structures contribute to the genomic instability associated with cancer.

QM/MM MD Simulations on the Enzymatic Pathway of the Human Flap Endonuclease (hFEN1) Elucidating Common Cleavage Pathways to RNase H Enzymes

Flap endonucleases (FENs) are nucleic acid hydrolyzing enzymes in charge of excising 5′-small DNA and RNA fragments (flaps) protruding from nucleic acid structures during the lagging strand DNA replication or the long-patch base excision repair (LP-BER) processes. In this work we report, for the first time, an atomistic and energetic rendering of the enzymatic catalysis promoted by the human FEN1. After reconstruction of a reactive hFEN/double strand (ds) DNA adduct we employed mixed quantum-classical (QM/MM) metadynamics and umbrella sampling free energy calculations, with the QM part treated with the AM1/d-PhoT Hamiltonian, to perform an extensive characterization of all possible reaction pathways underlying the enzymatic cycle. Our extensive investigation points to a most likely reaction pathway very similar to that recently proposed for ribonuclease H, in which the rate-determining step is the nucleophilic attack of a water to the scissile phosphate, which occurs concomitantly with its activation by t...

Binding polarity of RPA to telomeric sequences and influence of G-quadruplex stability.

Replication protein A (RPA) is a single-stranded DNA binding protein that plays an essential role in telomere maintenance. RPA binds to and unfolds G-quadruplex (G4) structures formed in telomeric DNA, thus facilitating lagging strand DNA replication and telomerase activity. To investigate the effect of G4 stability on the interactions with human RPA (hRPA), we used a combination of biochemical and biophysical approaches. Our data revealed an inverse relationship between G4 stability and ability of hRPA to bind to telomeric DNA; notably small G4 ligands that enhance G4 stability strongly impaired G4 unfolding by hRPA. To gain more insight into the mechanism of binding and unfolding of telomeric G4 structures by RPA, we carried out photo-crosslinking experiments to elucidate the spatial arrangement of the RPA subunits along the DNA strands. Our results showed that RPA1 and RPA2 are arranged from 5' to 3' along the unfolded telomeric G4, as already described for unstructured single-stranded DNA, while no contact is possible with RPA3 on this short oligonucleotide. In addition, these data are compatible with a 5' to 3' directionality in G4 unfolding by hRPA.

Effect of dNTP pool alterations on fidelity of leading and lagging strand DNA replication in E. coli

The fidelity with which organisms replicate their chromosomal DNA is of considerable interest. Detailed studies in the bacterium Escherichia coli have indicated that the fidelity of leading- and lagging-strand DNA replication is not the same, based on experiments in which the orientation of certain mutational targets on the chromosome was inverted relative to the movement of the replication fork: different mutation rates for several base-pair substitutions were observed depending on this orientation. While these experiments are indicative of differential replication fidelity in the two strands, a conclusion whether leading or lagging strand is the more accurate depends on knowledge of the primary mispairing error responsible for the base substitutions in question. A broad analysis of in vitro base-pairing preferences of DNA polymerases led us to propose that lagging-strand is the more accurate strand. In the present work, we present more direct in vivo evidence in support of this proposal. We determine the orientation dependence of mutant frequencies in ndk and dcd strains, which carry defined dNTP pool alterations. As these pool alterations lead to predictable effects on the array of possible mispairing errors, they mark the strands in which the observed errors occur. The combined results support the proposed higher accuracy of lagging-strand replication in E. coli.

Dna2 Is Involved in CA Strand Resection and Nascent Lagging Strand Completion at Native Yeast Telomeres

Post-replicational telomere end processing involves both extension by telomerase and resection to produce 3'-GT-overhangs that extend beyond the complementary 5'-CA-rich strand. Resection must be carefully controlled to maintain telomere length. At short de novo telomeres generated artificially by HO endonuclease in the G2 phase, we show that dna2-defective strains are impaired in both telomere elongation and sequential 5'-CA resection. At native telomeres in dna2 mutants, GT-overhangs do clearly elongate during late S phase but are shorter than in wild type, suggesting a role for Dna2 in 5'-CA resection but also indicating significant redundancy with other nucleases. Surprisingly, elimination of Mre11 nuclease or Exo1, which are complementary to Dna2 in resection of internal double strand breaks, does not lead to further shortening of GT-overhangs in dna2 mutants. A second step in end processing involves filling in of the CA-strand to maintain appropriate telomere length. We show that Dna2 is required for normal telomeric CA-strand fill-in. Yeast dna2 mutants, like mutants in DNA ligase 1 (cdc9), accumulate low molecular weight, nascent lagging strand DNA replication intermediates at telomeres. Based on this and other results, we propose that FEN1 is not sufficient and that either Dna2 or Exo1 is required to supplement FEN1 in maturing lagging strands at telomeres. Telomeres may be among the subset of genomic locations where Dna2 helicase/nuclease is essential for the two-nuclease pathway of primer processing on lagging strands.