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41.
42.
Giovanni Maga Barbara van Loon Emmanuele Crespan Giuseppe Villani Ulrich H��bscher 《The Journal of biological chemistry》2009,284(21):14267-14275
Abasic (AP) sites are very frequent and dangerous DNA lesions. Their
ability to block the advancement of a replication fork has been always viewed
as a consequence of their inhibitory effect on the DNA synthetic activity of
replicative DNA polymerases (DNA pols). Here we show that AP sites can also
affect the strand displacement activity of the lagging strand DNA pol δ,
thus preventing proper Okazaki fragment maturation. This block can be overcome
through a polymerase switch, involving the combined physical and functional
interaction of DNA pol β and Flap endonuclease 1. Our data identify a
previously unnoticed deleterious effect of the AP site lesion on normal cell
metabolism and suggest the existence of a novel repair pathway that might be
important in preventing replication fork stalling.Loss of purine and pyrimidine bases is a significant source of DNA damage
in prokaryotic and eukaryotic organisms. Abasic (apurinic and apyrimidinic)
lesions occur spontaneously in DNA; in eukaryotes it has been estimated that
about 104 depurination and 102 depyrimidation events
occur per genome per day. An equally important source of abasic DNA lesions
results from the action of DNA glycosylases, such as uracil glycosylase, which
excises uracil arising primarily from spontaneous deamination of cytosines
(1). Although most AP sites are
removed by the base excision repair
(BER)5 pathway, a
small fraction of lesions persists, and DNA with AP lesions presents a strong
block to DNA synthesis by replicative DNA polymerases (DNA pols)
(2,
3). Several studies have been
performed to address the effects of AP sites on the template DNA strand on the
synthetic activity of a variety of DNA pols. The major replicative enzyme of
eukaryotic cells, DNA pol δ, was shown to be able to bypass an AP
lesion, but only in the presence of the auxiliary factor proliferating cell
nuclear antigen (PCNA) and at a very reduced catalytic efficiency if compared
with an undamaged DNA template
(4). On the other hand, the
family X DNA pols β and λ were shown to bypass an AP site but in a
very mutagenic way (5). Recent
genetic evidence in Saccharomyces cerevisiae cells showed that DNA
pol δ is the enzyme replicating the lagging strand
(6). According to the current
model for Okazaki fragment synthesis
(7–9),
the action of DNA pol δ is not only critical for the extension of the
newly synthesized Okazaki fragment but also for the displacement of an RNA/DNA
segment of about 30 nucleotides on the pre-existing downstream Okazaki
fragment to create an intermediate Flap structure that is the target for the
subsequent action of the Dna2 endonuclease and the Flap endonuclease 1
(Fen-1). This process has the advantage of removing the entire RNA/DNA hybrid
fragment synthesized by the DNA pol α/primase, potentially containing
nucleotide misincorporations caused by the lack of a proofreading exonuclease
activity of DNA pol α/primase. This results in a more accurate copy
synthesized by DNA pol δ. The intrinsic strand displacement activity of
DNA pol δ, in conjunction with Fen-1, PCNA, and replication protein A
(RP-A), has been also proposed to be essential for the S phase-specific long
patch BER pathway (10,
11). Although it is clear that
an AP site on the template strand is a strong block for DNA pol
δ-dependent synthesis on single-stranded DNA, the functional
consequences of such a lesion on the ability of DNA pol δ to carry on
strand displacement synthesis have never been investigated so far. Given the
high frequency of spontaneous hydrolysis and/or cytidine deamination events,
any detrimental effect of an AP site on the strand displacement activity of
DNA pol δ might have important consequences both for lagging strand DNA
synthesis and for long patch BER. In this work, we addressed this issue by
constructing a series of synthetic gapped DNA templates with a single AP site
at different positions with respect to the downstream primer to be displaced
by DNA pol δ (see Fig.
1A). We show that an AP site immediately upstream of a
single- to double-strand DNA junction constitutes a strong block to the strand
displacement activity of DNA pol δ, even in the presence of RP-A and
PCNA. Such a block could be resolved only through a “polymerase
switch” involving the concerted physical and functional interaction of
DNA pol β and Fen-1. The closely related DNA pol λ could only
partially substitute for DNA pol β. Based on our data, we propose that
stalling of a replication fork by an AP site not only is a consequence of its
ability to inhibit nucleotide incorporation by the replicative DNA pols but
can also stem from its effects on strand displacement during Okazaki fragment
maturation. In summary, our data suggest the existence of a novel repair
pathway that might be important in preventing replication fork stalling and
identify a previously unnoticed deleterious effect of the AP site lesion on
normal cell metabolism.Open in a separate windowFIGURE 1.An abasic site immediately upstream of a double-stranded DNA region
inhibits the strand displacement activity of DNA polymerase δ. The
reactions were performed as described under “Experimental
Procedures.” A, schematic representation of the various DNA
templates used. The size of the resulting gaps is indicated in nt. The
position of the AP site on the 100-mer template strand is indicated relative
to the 3′ end. Base pairs in the vicinity of the lesion are indicated by
dashes. The size of the gaps (35–38 nt) is consistent with the
size of ssDNA covered by a single RP-A molecule, which has to be released
during Okazaki fragment synthesis when the DNA pol is approaching the
5′-end of the downstream fragment. When the AP site is covered by the
downstream terminator oligonucleotide (Gap-3 and Gap-1 templates) the
nucleotide placed on the opposite strand is C to mimic the situation generated
by spontaneous loss of a guanine or excision of an oxidized guanine, whereas
when the AP site is covered by the primer (nicked AP template), the nucleotide
placed on the opposite strand is A to mimic the most frequent incorporation
event occurring opposite an AP site. B, human PCNA was titrated in
the presence of 15 nm (lanes 2–4 and
10–12) or 30 nm (lanes 6–8 and
14–16) recombinant human four subunit DNA pol δ, on a
linear control (lanes 1–8) or a 38-nt gap control (lanes
9–16) template. Lanes 1, 5, 9, and 13, control
reactions in the absence of PCNA. C, human PCNA was titrated in the
presence of 60 nm DNA pol δ, on a linear AP (lanes
2–4) or 38-nt gap AP (lanes 6–9) template. Lanes
1 and 5, control reactions in the absence of PCNA. 相似文献
43.
Ramadan K Maga G Shevelev IV Villani G Blanco L Hübscher U 《Journal of molecular biology》2003,328(1):63-72
DNA polymerase lambda is a novel enzyme of the family X of DNA polymerases. The recent demonstration of an intrinsic 5'-deoxyribose-5'-phosphate lyase activity, a template/primer dependent polymerase activity, a distributive manner of DNA synthesis and sequence similarity to DNA polymerase beta suggested a novel beta-like enzyme. All these properties support a role of DNA polymerase lambda in base excision repair. On the other hand, the biochemical properties of the polymerisation activity of DNA polymerase lambda are still largely unknown. Here we give evidence that human DNA polymerase lambda has an intrinsic terminal deoxyribonucleotidyl transferase activity that preferentially adds pyrimidines onto 3'OH ends of DNA oligonucleotides. Furthermore, human DNA polymerase lambda efficiently elongates an RNA primer hybridized to a DNA template. These two novel properties of human DNA polymerase lambda might suggest additional roles for this enzyme in DNA replication and repair processes. 相似文献
44.
45.
Cell cycle-dependent dynamic association of cyclin/Cdk complexes with human DNA replication proteins
Frouin I Montecucco A Biamonti G Hübscher U Spadari S Maga G 《The EMBO journal》2002,21(10):2485-2495
We have previously described the isolation of a replication competent (RC) complex from calf thymus, containing DNA polymerase alpha, DNA polymerase delta and replication factor C. Here, we describe the isolation of the RC complex from nuclear extracts of synchronized HeLa cells, which contains DNA replication proteins associated with cell-cycle regulation factors like cyclin A, cyclin B1, Cdk2 and Cdk1. In addition, it contains a kinase activity and DNA polymerase activities able to switch from a distributive to a processive mode of DNA synthesis, which is dependent on proliferating cell nuclear antigen. In vivo cross-linking of proteins to DNA in synchronized HeLa cells demonstrates the association of this complex to chromatin. We show a dynamic association of cyclins/Cdks with the RC complex during the cell cycle. Indeed, cyclin A and Cdk2 associated with the complex in S phase, and cyclin B1 and Cdk1 were present exclusively in G(2)/M phase, suggesting that the activity, as well the localization, of the RC complex might be regulated by specific cyclin/Cdk complexes. 相似文献
46.
DNA polymerase beta was isolated from rat cortex neurons and characterised. Its properties were strikingly similar to those of other mammalian beta-polymerases. In adult rats, this was the major DNA polymerase occurring in neuronal nuclei, which contained no alpha-polymerase, 99.2% beta-polymerase and only 0.8% gamma-polymerase. Isolated neuronal nuclei of this developmental stage were shown to perform ultraviolet-induced repair DNA synthesis in vitro. Since beta-polymerase was virtually the exclusive DNA polymerase in these nuclei it was concluded that the beta enzyme was responsible for the observed DNA repair. This was further substantiated by demonstrating a virtually complete suppression of DNA repair in irradiated nuclei by 2',3'-dideoxyribosylthymine 5'-triphosphate (d2TTP), a potent beta-polymerase inhibitor. However, the presence of minute amounts of gamma-polymerase in neuronal nuclei and its susceptibility to d2TTP did not allow one to rule out an ancillary role of DNA polymerase gamma in DNA repair. In view of the similarity of the neuronal DNA polymerase beta with all other mammalian beta-polymerases it may be speculated that the ability to perform repair DNA synthesis is not unique to the neuronal enzyme but is a general function of all beta-polymerases. 相似文献
47.
Detection of the catalytic activities of DNA polymerases and their associated exonucleases following SDS-polyacrylamide gel electrophoresis. 总被引:37,自引:11,他引:26 下载免费PDF全文
A Spanos S G Sedgwick G T Yarranton U Hübscher G R Banks 《Nucleic acids research》1981,9(8):1825-1839
A method is described to detect DNA polymerases and nucleases in homogeneous or crude enzyme preparations after electrophoresis in SDS-polyacrylamide gels(2) containing the appropriate template or substrate. DNA polymerases are electrophoresed in a gel containing gapped calf thymus DNA and after a renaturation treatment, the gel is incubated in a reaction mixture in which one deoxyribonucleoside triphosphate is [alpha-32P]-labelled. Incorporation of radioactivity into DNA is detected at the vicinity of the polymerase band by autoradiography. An associated nuclease activity can be measured after electrophoresis in a gel containing 32P-labelled gapped DNA, when nucleolytic digestion is seen as a clear band in the resulting autoradiogram. The gels can subsequently be stained with Coomassie blue to establish identical molecular weights of polymerase, nuclease and protein bands. Applications of this technique are discussed. 相似文献
48.
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. 相似文献
49.
Electron microscopic analysis reveals that replication factor C is sequestered by single-stranded DNA. 总被引:1,自引:0,他引:1 下载免费PDF全文
R C Keller R Mossi G Maga R E Wellinger U Hübscher J M Sogo 《Nucleic acids research》1999,27(17):3433-3437
Replication factor C (RF-C) is a eukaryotic heteropentameric protein required for DNA replication and repair processes by loading proliferating cell nuclear antigen (PCNA) onto DNA in an ATP-dependent manner. Prior to loading PCNA, RF-C binds to DNA. This binding is thought to be restricted to a specific DNA structure, namely to a primer/template junction. Using the electron microscope we have examined the affinity of human heteropentameric RF-C and the DNA-binding region within the large subunit of RF-C from Drosophila melanogaster (dRF-Cp140) to heteroduplex DNA. The electron microscopic data indicate that both human heteropentameric RF-C and the DNA-binding region within dRF-Cp140 are sequestered by single-stranded DNA. No preferential affinity for the 3' or 5' transition points from single- to double-stranded DNA was evident. 相似文献
50.
A novel proteomic approach identifies new interaction partners for proliferating cell nuclear antigen 总被引:2,自引:0,他引:2
Meslet-Cladiére L Norais C Kuhn J Briffotaux J Sloostra JW Ferrari E Hübscher U Flament D Myllykallio H 《Journal of molecular biology》2007,372(5):1137-1148
During DNA replication and repair, many proteins bind to and dissociate in a highly specific and ordered manner from proliferating cell nuclear antigen (PCNA). We describe a combined approach of in silico searches at the genome level and combinatorial peptide synthesis to investigate the binding properties of hundreds of short PCNA-interacting peptides (PIP-peptides) to archaeal and eukaryal PCNAs. Biological relevance of our combined approach was demonstrated by identification an inactive complex of Pyrococcus abyssi ribonuclease HII with PCNA. Furthermore we show that PIP-peptides interact with PCNA largely in a sequence independent manner. Our experimental approach also identified many so far unidentified PCNA interacting peptides in a number of human proteins. 相似文献