A Role for Human DNA Polymerase λ in Alternative Lengthening of Telomeres.

Telomerase negative cancer cell types use the Alternative Lengthening of Telomeres (ALT) pathway to elongate telomeres ends. Here, we show that silencing human DNA polymerase (Pol λ) in ALT cells represses ALT activity and induces telomeric stress. In addition, replication stress in the absence of Pol λ, strongly affects the survival of ALT cells. In vitro, Pol λ can promote annealing of even a single G-rich telomeric repeat to its complementary strand and use it to prime DNA synthesis. The noncoding telomeric repeat containing RNA TERRA and replication protein A negatively regulate this activity, while the Protection of Telomeres protein 1 (POT1)/TPP1 heterodimer stimulates Pol λ. Pol λ associates with telomeres and colocalizes with TPP1 in cells. In summary, our data suggest a role of Pol λ in the maintenance of telomeres by the ALT mechanism.

Ribonucleotide incorporation by human DNA polymerase η impacts translesion synthesis and RNase H2 activity.

Ribonucleotides (rNs) incorporated in the genome by DNA polymerases (Pols) are removed by RNase H2. Cytidine and guanosine preferentially accumulate over the other rNs. Here we show that human Pol η can incorporate cytidine monophosphate (rCMP) opposite guanine, 8-oxo-7,8-dihydroguanine, 8-methyl-2΄-deoxyguanosine and a cisplatin intrastrand guanine crosslink (cis-PtGG), while it cannot bypass a 3-methylcytidine or an abasic site with rNs as substrates. Pol η is also capable of synthesizing polyribonucleotide chains, and its activity is enhanced by its auxiliary factor DNA Pol δ interacting protein 2 (PolDIP2). Human RNase H2 removes cytidine and guanosine less efficiently than the other rNs and incorporation of rCMP opposite DNA lesions further reduces the efficiency of RNase H2. Experiments with XP-V cell extracts indicate Pol η as the major basis of rCMP incorporation opposite cis-PtGG. These results suggest that translesion synthesis by Pol η can contribute to the accumulation of rCMP in the genome, particularly opposite modified guanines.

Living on the Edge: DNA Polymerase Lambda between Genome Stability and Mutagenesis.

In human cells, only four DNA polymerases (pols) are necessary and sufficient for the duplication of the genetic information. However, more than a dozen DNA pols are required to maintain its integrity. Such a high degree of specialization makes DNA repair pols able to cope with specific lesions or repair pathways. On the other hand, the same DNA pols can have partially overlapping roles, which could result in possible conflicts of functions, if the DNA pols are not properly regulated. DNA pol λ is a typical example of such an enzyme. It is a multifunctional enzyme, endowed with special structural and biochemical properties, which make it capable of participating in different DNA repair pathways such as base excision repair, nonhomologous end joining, and translesion synthesis. However, when mutated or deregulated, DNA pol λ can also be a source of genetic instability. Its multiple roles in DNA damage tolerance and its ability in promoting tumor progression make it also a possible target for novel anticancer approaches.

DNA polymerase δ-interacting protein 2 is a processivity factor for DNA polymerase λ during 8-oxo-7,8-dihydroguanine bypass.

The bypass of DNA lesions by the replication fork requires a switch between the replicative DNA polymerase (Pol) and a more specialized translesion synthesis (TLS) Pol to overcome the obstacle. DNA Pol δ-interacting protein 2 (PolDIP2) has been found to physically interact with Pol η, Pol ζ, and Rev1, suggesting a possible role of PolDIP2 in the TLS reaction. However, the consequences of PolDIP2 interaction on the properties of TLS Pols remain unknown. Here, we analyzed the effects of PolDIP2 on normal and TLS by five different human specialized Pols from three families: Pol δ (family B), Pol η and Pol ι (family Y), and Pol λ and Pol ß (family X). Our results show that PolDIP2 also physically interacts with Pol λ, which is involved in the correct bypass of 8-oxo-7,8-dihydroguanine (8-oxo-G) lesions. This interaction increases both the processivity and catalytic efficiency of the error-free bypass of a 8-oxo-G lesion by both Pols η and λ, but not by Pols ß or ι. Additionally, we provide evidence that PolDIP2 stimulates Pol δ without affecting its fidelity, facilitating the switch from Pol δ to Pol λ during 8-oxo-G TLS. PolDIP2 stimulates Pols λ and η mediated bypass of other common DNA lesions, such as abasic sites and cyclobutane thymine dimers. Finally, PolDIP2 silencing increases cell sensitivity to oxidative stress and its effect is further potentiated in a Pol λ deficient background, suggesting that PolDIP2 is an important mediator for TLS.

Regulation of human MutYH DNA glycosylase by the E3 ubiquitin ligase mule.

Oxidation of DNA is a frequent and constantly occurring event. One of the best characterized oxidative DNA lesions is 7,8-dihydro-8-oxoguanine (8-oxo-G). It instructs most DNA polymerases to preferentially insert an adenine (A) opposite 8-oxo-G instead of the appropriate cytosine (C) thus showing miscoding potential. The MutY DNA glycosylase homologue (MutYH) recognizes A:8-oxo-G mispairs and removes the mispaired A giving way to the canonical base excision repair that ultimately restores undamaged guanine (G). Here we characterize for the first time in detail a posttranslational modification of the human MutYH DNA glycosylase. We show that MutYH is ubiquitinated in vitro and in vivo by the E3 ligase Mule between amino acids 475 and 535. Mutation of five lysine residues in this region significantly stabilizes MutYH, suggesting that these are the target sites for ubiquitination. The endogenous MutYH protein levels depend on the amount of expressed Mule. Furthermore, MutYH and Mule physically interact. We found that a ubiquitination-deficient MutYH mutant shows enhanced binding to chromatin. The mutation frequency of the ovarian cancer cell line A2780, analyzed at the HPRT locus can be increased upon oxidative stress and depends on the MutYH levels that are regulated by Mule. This reflects the importance of tightly regulated MutYH levels in the cell. In summary our data show that ubiquitination is an important regulatory mechanism for the essential MutYH DNA glycosylase in human cells.

Agnoprotein of polyomavirus BK interacts with proliferating cell nuclear antigen and inhibits DNA replication.

BACKGROUND: The human polyomavirus BK expresses a 66 amino-acid peptide referred to as agnoprotein. Though mutants lacking agnoprotein are severely reduced in producing infectious virions, the exact function of this peptide remains incompletely understood. To elucidate the function of agnoprotein, we searched for novel cellular interaction partners. METHODS: Yeast-two hybrid assay was performed with agnoprotein as bait against human kidney and thymus libraries. The interaction between agnoprotein and putative partners was further examined by GST pull down, co-immunoprecipitation, and fluorescence resonance energy transfer studies. Biochemical and biological studies were performed to examine the functional implication of the interaction of agnoprotein with cellular target proteins. RESULTS: Proliferating cell nuclear antigen (PCNA), which acts as a processivity factor for DNA polymerase δ, was identified as an interaction partner. The interaction between agnoprotein and PCNA is direct and occurs also in human cells. Agnoprotein exerts an inhibitory effect on PCNA-dependent DNA synthesis in vitro and reduces cell proliferation when ectopically expressed. Overexpression of PCNA restores agnoprotein-mediated inhibition of cell proliferation. CONCLUSION: Our data suggest that PCNA is a genuine interaction partner of agnoprotein and the inhibitory effect on PCNA-dependent DNA synthesis by the agnoprotein may play a role in switching off (viral) DNA replication late in the viral replication cycle when assembly of replicated genomes and synthesized viral capsid proteins occurs.

Mammalian base excision repair: the forgotten archangel.

Base excision repair (BER) is a frontline repair system that is responsible for maintaining genome integrity and thus preventing premature aging, cancer and many other human diseases by repairing thousands of DNA lesions and strand breaks continuously caused by endogenous and exogenous mutagens. This fundamental and essential function of BER not only necessitates tight control of the continuous availability of basic components for fast and accurate repair, but also requires temporal and spatial coordination of BER and cell cycle progression to prevent replication of damaged DNA. The major goal of this review is to critically examine controversial and newly emerging questions about mammalian BER pathways, mechanisms regulating BER capacity, BER responses to DNA damage and their links to checkpoint control of DNA replication.

Silencing of human DNA polymerase λ causes replication stress and is synthetically lethal with an impaired S phase checkpoint.

Human DNA polymerase (pol) λ functions in base excision repair and non-homologous end joining. We have previously shown that DNA pol λ is involved in accurate bypass of the two frequent oxidative lesions, 7,8-dihydro-8-oxoguanine and 1,2-dihydro-2-oxoadenine during the S phase. However, nothing is known so far about the relationship of DNA pol λ with the S phase DNA damage response checkpoint. Here, we show that a knockdown of DNA pol λ, but not of its close homologue DNA pol ß, results in replication fork stress and activates the S phase checkpoint, slowing S phase progression in different human cancer cell lines. We furthermore show that DNA pol λ protects cells from oxidative DNA damage and also functions in rescuing stalled replication forks. Its absence becomes lethal for a cell when a functional checkpoint is missing, suggesting a DNA synthesis deficiency. Our results provide the first evidence, to our knowledge, that DNA pol λ is required for cell cycle progression and is functionally connected to the S phase DNA damage response machinery in cancer cells.

A switch between DNA polymerases δ and λ promotes error-free bypass of 8-oxo-G lesions.

7,8-Dihydro-8-oxoguanine (8-oxo-G) is a highly abundant and mutagenic lesion. Replicative DNA polymerases (pols) are slowed down at 8-oxo-G and insert both correct cytosine (C) and incorrect adenine (A) opposite 8-oxo-G, but they preferentially extend A:8-oxo-G mispairs. Nevertheless, 8-oxo-G bypass is fairly accurate in vivo. Thus, the question how correct bypass of 8-oxo-G lesions is accomplished despite the poor extension of C:8-oxo-G base pairs by replicative pols remains unanswered. Here we show that replicative pol δ pauses in front of 8-oxo-G and displays difficulties extending from correct C:8-oxo-G in contrast to extension from incorrect A:8-oxo-G. This leads to stalling of pol δ at 8-oxo-G after incorporation of correct C. This stalling at C:8-oxo-G can be overcome by a switch from pol δ to pols λ, ß, or η, all of which are able to assist pol δ in 8-oxo-G bypass by translesion synthesis (TLS). Importantly, however, only pol λ selectively catalyzes the correct TLS past 8-oxo-G, whereas pols ß and η show no selectivity and even preferentially enhance incorrect TLS. The selectivity of pol λ to promote the correct bypass depends on its N-terminal domain. Furthermore, pol λ(-/-) mouse embryonic fibroblast extracts display reduced 8-oxo-G TLS. Finally, the correct bypass of 8-oxo-G in gapped plasmids in mouse embryonic fibroblasts and HeLa cells is promoted in the presence of pol λ. Our findings suggest that even though 8-oxo-G is not a blocking lesion per se, correct replication over 8-oxo-G is promoted by a pol switch between pols δ and λ.

Regulation of oxidative DNA damage repair by DNA polymerase λ and MutYH by cross-talk of phosphorylation and ubiquitination.

It is of pivotal importance for genome stability that repair DNA polymerases (Pols), such as Pols λ and ß, which all exhibit considerably reduced fidelity when replicating undamaged DNA, are tightly regulated, because their misregulation could lead to mutagenesis. Recently, we found that the correct repair of the abundant and highly miscoding oxidative DNA lesion 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxo-G) is performed by an accurate repair pathway that is coordinated by the MutY glycosylase homologue (MutYH) and Pol λ in vitro and in vivo. Pol λ is phosphorylated by Cdk2/cyclinA in late S and G2 phases of the cell cycle, promoting Pol λ stability by preventing it from being targeted for proteasomal degradation by ubiquitination. However, it has remained a mystery how the levels of Pol λ are controlled, how phosphorylation promotes its stability, and how the engagement of Pol λ in active repair complexes is coordinated. Here, we show that the E3 ligase Mule mediates the degradation of Pol λ and that the control of Pol λ levels by Mule has functional consequences for the ability of mammalian cells to deal with 8-oxo-G lesions. Furthermore, we demonstrate that phosphorylation of Pol λ by Cdk2/cyclinA counteracts its Mule-mediated degradation by promoting recruitment of Pol λ to chromatin into active 8-oxo-G repair complexes through an increase in Pol λ's affinity to chromatin-bound MutYH. Finally, MutYH appears to promote the stability of Pol λ by binding it to chromatin. In contrast, Pol λ not engaged in active repair on chromatin is subject for proteasomal degradation.

The human Tim-Tipin complex interacts directly with DNA polymerase epsilon and stimulates its synthetic activity.

The Tim-Tipin complex plays an important role in the S phase checkpoint and replication fork stability in metazoans, but the molecular mechanism underlying its biological function is poorly understood. Here, we present evidence that the recombinant human Tim-Tipin complex (and Tim alone) markedly enhances the synthetic activity of DNA polymerase ε. In contrast, no significant effect on the synthetic ability of human DNA polymerase α and δ by Tim-Tipin was observed. Surface plasmon resonance measurements and co-immunoprecipitation experiments revealed that recombinant DNA polymerase ε directly interacts with either Tim or Tipin. In addition, the results of DNA band shift assays suggest that the Tim-Tipin complex (or Tim alone) is able to associate with DNA polymerase ε bound to a 40-/80-mer DNA ligand. Our results are discussed in view of the molecular dynamics at the human DNA replication fork.

Microhomology-mediated DNA strand annealing and elongation by human DNA polymerases λ and ß on normal and repetitive DNA sequences.

'Classical' non-homologous end joining (NHEJ), dependent on the Ku70/80 and the DNA ligase IV/XRCC4 complexes, is essential for the repair of DNA double-strand breaks. Eukaryotic cells possess also an alternative microhomology-mediated end-joining (MMEJ) mechanism, which is independent from Ku and DNA ligase 4/XRCC4. The components of the MMEJ machinery are still largely unknown. Family X DNA polymerases (pols) are involved in the classical NHEJ pathway. We have compared in this work, the ability of human family X DNA pols ß, λ and µ, to promote the MMEJ of different model templates with terminal microhomology regions. Our results reveal that DNA pol λ and DNA ligase I are sufficient to promote efficient MMEJ repair of broken DNA ends in vitro, and this in the absence of auxiliary factors. However, DNA pol ß, not λ, was more efficient in promoting MMEJ of DNA ends containing the (CAG)n triplet repeat sequence of the human Huntingtin gene, leading to triplet expansion. The checkpoint complex Rad9/Hus1/Rad1 promoted end joining by DNA pol λ on non-repetitive sequences, while it limited triplet expansion by DNA pol ß. We propose a possible novel role of DNA pol ß in MMEJ, promoting (CAG)n triplet repeats instability.

Involvement of Werner syndrome protein in MUTYH-mediated repair of oxidative DNA damage.

Reactive oxygen species constantly generated as by-products of cellular metabolism readily attack genomic DNA creating mutagenic lesions such as 7,8-dihydro-8-oxo-guanine (8-oxo-G) that promote aging. 8-oxo-G:A mispairs arising during DNA replication are eliminated by base excision repair initiated by the MutY DNA glycosylase homologue (MUTYH). Here, by using formaldehyde crosslinking in mammalian cell extracts, we demonstrate that the WRN helicase/exonuclease defective in the premature aging disorder Werner syndrome (WS) is recruited to DNA duplex containing an 8-oxo-G:A mispair in a manner dependent on DNA polymerase λ (Polλ) that catalyzes accurate DNA synthesis over 8-oxo-G. Similarly, by immunofluorescence, we show that Polλ is required for accumulation of WRN at sites of 8-oxo-G lesions in human cells. Moreover, we show that nuclear focus formation of WRN and Polλ induced by oxidative stress is dependent on ongoing DNA replication and on the presence of MUTYH. Cell viability assays reveal that depletion of MUTYH suppresses the hypersensitivity of cells lacking WRN and/or Polλ to oxidative stress. Biochemical studies demonstrate that WRN binds to the catalytic domain of Polλ and specifically stimulates DNA gap filling by Polλ over 8-oxo-G followed by strand displacement synthesis. Our results suggest that WRN promotes long-patch DNA repair synthesis by Polλ during MUTYH-initiated repair of 8-oxo-G:A mispairs.

A new proofreading mechanism for lesion bypass by DNA polymerase-λ.

Replicative DNA polymerases (DNA pols) increase their fidelity by removing misincorporated nucleotides with their 3' → 5' exonuclease activity. Exonuclease activity reduces translesion synthesis (TLS) efficiency and TLS DNA pols lack 3' → 5' exonuclease activity. Here we show that physiological concentrations of pyrophosphate (PP(i)) activate the pyrophosphorolytic activity by DNA pol-λ, allowing the preferential excision of the incorrectly incorporated A opposite a 7,8-dihydro-8-oxoguanine lesion, or T opposite a 6-methyl-guanine, with respect to the correct C. This is the first example of an alternative proofreading mechanism used during TLS.

8-oxo-guanine bypass by human DNA polymerases in the presence of auxiliary proteins.

Specialized DNA polymerases (DNA pols) are required for lesion bypass in human cells. Auxiliary factors have an important, but so far poorly understood, role. Here we analyse the effects of human proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A) on six different human DNA pols--belonging to the B, Y and X classes--during in vitro bypass of different lesions. The mutagenic lesion 8-oxo-guanine (8-oxo-G) has high miscoding potential. A major and specific effect was found for 8-oxo-G bypass with DNA pols lambda and eta. PCNA and RP-A allowed correct incorporation of dCTP opposite a 8-oxo-G template 1,200-fold more efficiently than the incorrect dATP by DNA pol lambda, and 68-fold by DNA pol eta, respectively. Experiments with DNA-pol-lambda-null cell extracts suggested an important role for DNA pol lambda. On the other hand, DNA pol iota, together with DNA pols alpha, delta and beta, showed a much lower correct bypass efficiency. Our findings show the existence of an accurate mechanism to reduce the deleterious consequences of oxidative damage and, in addition, point to an important role for PCNA and RP-A in determining a functional hierarchy among different DNA pols in lesion bypass.

In vitro gap-directed translesion DNA synthesis of an abasic site involving human DNA polymerases epsilon, lambda, and beta.

DNA polymerase (pol) ε is thought to be the leading strand replicase in eukaryotes, whereas pols λ and ß are believed to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol ε is halted by an abasic site (apurinic/apyrimidinic (AP) site). In this study, we present in vitro evidence that human pols λ, ß, and η can perform translesion synthesis (TLS) of an AP site in the presence of pol ε, likely by initiating the 3'OHs created at the lesion by the arrested pol ε. However, in the case of pols λ and ß, this TLS requires the presence of a DNA gap downstream from the product synthesized by the pol ε, and the optimal gap for efficient TLS is different for the two polymerases. The presence of gaps did not affect the TLS capacity of human pol η. Characterization of the reaction products showed that pol ß inserted dAMP opposite the AP site, whereas gap filling synthesis by pol λ resulted in single or double deletions opposite the lesion. The synthesis up to the AP site by pol ε and the subsequent TLS by pols λ and ß are not influenced by human processivity factor proliferating cell nuclear antigen and human single-stranded DNA-binding protein replication protein A. The bypass capacity of pol λ at the AP site is greatly reduced when a truncated form of the enzyme, which has lost the BRCA1 C-terminal and proline-rich domains, is used. Collectively, our in vitro results support the existence of a mechanism of gap-directed TLS at an AP site involving a switch between the replicative pol ε and the repair pols λ and ß.

An 8-oxo-guanine repair pathway coordinated by MUTYH glycosylase and DNA polymerase lambda.

Reactive oxygen species (ROS) interact with DNA, frequently generating highly mutagenic 7,8-dihydro-8-oxoguanine (8-oxo-G) lesions. Replicative DNA polymerases (pols) often misincorporate adenine opposite 8-oxo-G. The subsequent repair mechanism allowing the removal of adenine and formation of C:8-oxo-G base pair is essential to prevent C:G to A:T transversion mutations. Here, we show by immunofluorescence experiments, in cells exposed to ROS, the involvement of MutY glycosylase homologue (MUTYH) and DNA pol lambda in the repair of A:8-oxo-G mispairs. We observe specific recruitment of MUTYH, DNA pol lambda, proliferating cell nuclear antigen (PCNA), flap endonuclease 1 (FEN1) and DNA ligases I and III from human cell extracts to A:8-oxo-G DNA, but not to undamaged DNA. Using purified human proteins and a DNA template, we reconstitute the full pathway for the faithful repair of A:8-oxo-G mispairs involving MUTYH, DNA pol lambda, FEN1, and DNA ligase I. These results reveal a cellular response pathway to ROS, important to sustain genomic stability and modulate carcinogenesis.

Generation of a mono-ubiquitinated PCNA mimic by click chemistry.

Genotoxic stress results in more than 50 000 damaged DNA sites per cell per day. During DNA replication, processive high-fidelity DNA polymerases generally stall at DNA lesions and have to be displaced by translesion synthesis DNA polymerases, which are able to bypass the lesion. This switch is mediated by mono-ubiquitination of the processivity factor proliferating cell nuclear antigen (PCNA). To further investigate the regulation of the DNA polymerase exchange, we developed an easy and efficient method to synthesize site-specifically mono-ubiquitinated PCNA by click chemistry. By incorporating artificial amino acids that carry an azide (Aha) or an alkyne (Plk) in their side chains, into ubiquitin (Ub) and PCNA, respectively, we were able to link the two proteins site-specifically by the Cu(I) -catalyzed azide-alkyne cycloaddition. Finally, we show that the synthetic PCNA-Ub is able to stimulate DNA synthesis by DNA polymerase δ, and that DNA polymerase η has a higher affinity for PCNA-Ub than to PCNA.

Effect of 8-oxoguanine and abasic site DNA lesions on in vitro elongation by human DNA polymerase in the presence of replication protein A and proliferating-cell nuclear antigen.

DNA pol (polymerase) is thought to be the leading strand replicase in eukaryotes. In the present paper, we show that human DNA pol can efficiently bypass an 8-oxo-G (7,8-dihydro-8-oxoguanine) lesion on the template strand by inserting either dCMP or dAMP opposite to it, but it cannot bypass an abasic site. During replication, DNA pols associate with accessory proteins that may alter their bypass ability. We investigated the role of the human DNA sliding clamp PCNA (proliferating-cell nuclear antigen) and of the human single-stranded DNA-binding protein RPA (replication protein A) in the modulation of the DNA synthesis and translesion capacity of DNA pol . RPA inhibited the elongation by human DNA pol on templates annealed to short primers. PCNA did not influence the elongation by DNA pol and had no effect on inhibition of elongation caused by RPA. RPA inhibition was considerably reduced when the length of the primers was increased. On templates bearing the 8-oxo-G lesion, this inhibitory effect was more pronounced on DNA replication beyond the lesion, suggesting that RPA may prevent extension by DNA pol after incorporation opposite an 8-oxo-G. Neither PCNA nor RPA had any effect on the inability of DNA pol to replicate past the AP site, independent of the primer length.

Replication protein A and proliferating cell nuclear antigen coordinate DNA polymerase selection in 8-oxo-guanine repair.

The adenine misincorporated by replicative DNA polymerases (pols) opposite 7,8-dihydro-8-oxoguanine (8-oxo-G) is removed by a specific glycosylase, leaving the lesion on the DNA. Subsequent incorporation of C opposite 8-oxo-G on the resulting 1-nt gapped DNA is essential for the removal of the 8-oxo-G to prevent G-C to T-A transversion mutations. By using model DNA templates, purified DNA pols beta and lambda and knockout cell extracts, we show here that the auxiliary proteins replication protein A and proliferating cell nuclear antigen act as molecular switches to activate the DNA pol lambda- dependent highly efficient and faithful repair of A:8-oxo-G mismatches in human cells and to repress DNA pol beta activity. By using an immortalized human fibroblast cell line that has the potential to induce cancer in mice, we show that the development of a tumoral phenotype in these cells correlated with a differential expression of DNA pols lambda and beta.