Biomedical subjects

Alan R Lehmann

Publications and source records attributed to Alan R Lehmann.

34 records · Page 2Linked to original sources

PubMed2005

Transcription-associated breaks in xeroderma pigmentosum group D cells from patients with combined features of xeroderma pigmentosum and Cockayne syndrome.

Defects in the XPD gene can result in several clinical phenotypes, including xeroderma pigmentosum (XP), trichothiodystrophy, and, less frequently, the combined phenotype of XP and Cockayne syndrome (XP-D/CS). We previously showed that in cells from two XP-D/CS patients, breaks were introduced into cellular DNA on exposure to UV damage, but these breaks were not at the sites of the damage. In the present work, we show that three further XP-D/CS patients show the same peculiar breakage phenomenon. We show that these breaks can be visualized inside the cells by immunofluorescence using antibodies to either gamma-H2AX or poly-ADP-ribose and that they can be generated by the introduction of plasmids harboring methylation or oxidative damage as well as by UV photoproducts. Inhibition of RNA polymerase II transcription by four different inhibitors dramatically reduced the number of UV-induced breaks. Furthermore, the breaks were dependent on the nucleotide excision repair (NER) machinery. These data are consistent with our hypothesis that the NER machinery introduces the breaks at sites of transcription initiation. During transcription in UV-irradiated XP-D/CS cells, phosphorylation of the carboxy-terminal domain of RNA polymerase II occurred normally, but the elongating form of the polymerase remained blocked at lesions and was eventually degraded.

Cockayne Syndrome↗

PubMed2004

Localisation of human Y-family DNA polymerase kappa: relationship to PCNA foci.

DNA polymerases of the Y-family are involved in translesion DNA synthesis past different types of DNA damage. Previous work has shown that DNA polymerases eta and iota are localised in replication factories during S phase, where they colocalise one-to-one with PCNA. Cells with factories containing these polymerases accumulate after treatment with DNA damaging agents because replication forks are stalled at sites of damage. We now show that DNA polymerase kappa (pol(kappa)) has a different localisation pattern. Although, like the other Y-family polymerases, it is exclusively localised in the nucleus, pol(kappa) is found in replication foci in only a small proportion of S-phase cells. It does not colocalise in those foci with proliferating cell nuclear antigen (PCNA) in the majority of cells. This reduced number of cells with pol(kappa) foci, when compared with those containing pol(eta) foci, is observed both in untreated cells and in cells treated with hydroxyurea, UV irradiation or benzo[a]pyrene. The C-terminal 97 amino acids of pol(kappa)are sufficient for this limited localisation into nuclear foci, and include a C2HC zinc finger, bipartite nuclear localisation signal and putative PCNA binding site.

Amino Acid Sequence↗

PubMed2004

Co-localization in replication foci and interaction of human Y-family members, DNA polymerase pol eta and REVl protein.

The progress of replicative DNA polymerases along the replication fork may be impeded by the presence of lesions in the genome. One way to circumvent such hurdles involves the recruitment of specialized DNA polymerases that perform limited incorporation of nucleotides in the vicinity of the damaged site. This process entails DNA polymerase switch between replicative and specialized DNA polymerases. Five eukaryotic proteins can carry out translesion synthesis (TLS) of damaged DNA in vitro, DNA polymerases zeta, eta, iota, and kappa, and REV1. To identify novel proteins that interact with hpol eta, we performed a yeast two-hybrid screen. In this paper, we show that hREV1 interacts with hpol eta as well as with hpol kappa and poorly with hpol iota. Furthermore, cellular localization analysis demonstrates that hREV1 is present, with hpol eta in replication factories at stalled replication forks and is tightly associated with nuclear structures. This hREV1 nuclear localization occurs independently of the presence of hpol eta. Taken together, our data suggest a central role for hREV1 as a scaffold that recruits DNA polymerases involved in TLS.

Base Sequence↗

PubMed2004

Proliferating cell nuclear antigen-dependent coordination of the biological functions of human DNA polymerase iota.

Y-family DNA polymerases are believed to facilitate the replicative bypass of damaged DNA in a process commonly referred to as translesion synthesis. With the exception of DNA polymerase eta (poleta), which is defective in humans with the Xeroderma pigmentosum variant (XP-V) phenotype, little is known about the cellular function(s) of the remaining human Y-family DNA polymerases. We report here that an interaction between human DNA polymerase iota (poliota) and the proliferating cell nuclear antigen (PCNA) stimulates the processivity of poliota in a template-dependent manner in vitro. Mutations in one of the putative PCNA-binding motifs (PIP box) of poliota or the interdomain connector loop of PCNA diminish the binding between poliota and PCNA and concomitantly reduce PCNA-dependent stimulation of poliota activity. Furthermore, although retaining its capacity to interact with poleta in vivo, the poliota-PIP box mutant fails to accumulate in replication foci. Thus, PCNA, acting as both a scaffold and a modulator of the different activities involved in replication, appears to recruit and coordinate replicative and translesion DNA synthesis polymerases to ensure genome integrity.

Amino Acid Sequence↗

PubMed2004

Ubiquitination of PCNA and the polymerase switch in human cells.

Replicative DNA polymerases are blocked by damage in the template DNA. To get past this damage, the cell employs specialized translesion synthesis (TLS) polymerases, which have reduced stringency and are able to bypass different lesions. For example, DNA polymerase eta (poleta) is able to carry out TLS past UV-induced cyclobutane pyrimidine dimers. How does the cell bring about the switch from replicative to TLS polymerase? We have shown that, in human cells, when the replication machinery is blocked at DNA damage, PCNA, the sliding clamp required for DNA replication, is mono-ubiquitinated and that this modified form of PCNA has increased affinity for poleta. This provides a mechanism for the polymerase switch. In this Extra-View, we discuss the possible signals that might trigger ubiquitination of PCNA, whether PCNA becomes de-ubiquitinated after TLS has been accomplished and the role of the hREV1 protein in TLS. We point out some apparent differences between mechanisms in Saccharomyces cerevisiae and human cells.

DNA Replication↗

PubMed2004

Interaction of human DNA polymerase eta with monoubiquitinated PCNA: a possible mechanism for the polymerase switch in response to DNA damage.

Most types of DNA damage block replication fork progression during DNA synthesis because replicative DNA polymerases are unable to accommodate altered DNA bases in their active sites. To overcome this block, eukaryotic cells employ specialized translesion synthesis (TLS) polymerases, which can insert nucleotides opposite damaged bases. In particular, TLS by DNA polymerase eta (poleta) is the major pathway for bypassing UV photoproducts. How the cell switches from replicative to TLS polymerase at the site of blocked forks is unknown. We show that, in human cells, PCNA becomes monoubiquitinated following UV irradiation of the cells and that this is dependent on the hRad18 protein. Monoubiquitinated PCNA but not unmodified PCNA specifically interacts with poleta, and we have identified two motifs in poleta that are involved in this interaction. Our findings provide an attractive mechanism by which monoubiquitination of PCNA might mediate the polymerase switch.

Chromatin↗

PubMed2004

DNA polymerases eta and iota.

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Animals↗

PubMed2004

Maintaining integrity.

Research on genome stability and integrity now extends far beyond the biochemistry of DNA repair to encompass signal transduction pathways that span numerous aspects of cellular life. Derailed genomic integrity pathways can result in debilitating genetic disorders, premature ageing, predisposition to cancer and degenerative conditions. Current progress in this rapidly expanding field was the subject of an EMBO workshop, Maintenance of Genomic Integrity, that took place in June 2004 in Galway, Ireland. Top

Animals↗

PubMed2003

Role of DNA polymerase eta in the UV mutation spectrum in human cells.

In humans, inactivation of the DNA polymerase eta gene (pol eta) results in sunlight sensitivity and causes the cancer-prone xeroderma pigmentosum variant syndrome (XP-V). Cells from XP-V individuals have a reduced capacity to replicate UV-damaged DNA and show hypermutability after UV exposure. Biochemical assays have demonstrated the ability of pol eta to bypass cis-syn-cyclobutane thymine dimers, the most common lesion generated in DNA by UV. In most cases, this bypass is error-free. To determine the actual requirement of pol eta in vivo, XP-V cells (XP30RO) were complemented by the wild type pol eta gene. We have used two pol eta-corrected clones to study the in vivo characteristics of mutations produced by DNA polymerases during DNA synthesis of UV-irradiated shuttle vectors transfected into human host cells, which had or had not been exposed previously to UV radiation. The functional complementation of XP-V cells by pol eta reduced the mutation frequencies both at CG and TA base pairs and restored UV mutagenesis to a normal level. UV irradiation of host cells prior to transfection strongly increased the mutation frequency in undamaged vectors and, in addition, especially in the pol eta-deficient XP30RO cells at 5'-TT sites in UV-irradiated plasmids. These results clearly show the protective role of pol eta against UV-induced lesions and the activation by UV of pol eta-independent mutagenic processes.

Base Sequence↗

PubMed2003

Localization of DNA polymerases eta and iota to the replication machinery is tightly co-ordinated in human cells.

Y-family DNA polymerases can replicate past a variety of damaged bases in vitro but, with the exception of DNA polymerase eta (poleta), which is defective in xeroderma pigmentosum variants, there is little information on the functions of these polymerases in vivo. Here, we show that DNA polymerase iota (poliota), like poleta, associates with the replication machinery and accumulates at stalled replication forks following DNA-damaging treatment. We show that poleta and poliota foci form with identical kinetics and spatial distributions, suggesting that localization of these two polymerases is tightly co-ordinated within the nucleus. Furthermore, localization of poliota in replication foci is largely dependent on the presence of poleta. Using several different approaches, we demonstrate that poleta and poliota interact with each other physically and that the C-terminal 224 amino acids of poliota are sufficient for both the interaction with poleta and accumulation in replication foci. Our results provide strong evidence that poleta targets poliota to the replication machinery, where it may play a general role in maintaining genome integrity as well as participating in translesion DNA synthesis.

Animals↗

PubMed2003

DNA repair-deficient diseases, xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy.

Xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD) are genetic disorders with very different clinical features, but all associated with defects in nucleotide excision repair. Defects in the XPA or XPC genes confer sensitivity to UV carcinogenesis in both humans and mice, but only XPA(-/-) mice have increased acute responses to UV exposure, whereas XPC(-/-) mice are normal in this respect. Both XPE and XPF proteins have functions separate from their role in NER, but the exact nature of these functions has not yet been established. The CSA and CSB genes responsible for CS are both components of complexes associated with RNA polymerase II and their role is thought to be in assisting polII in dealing with transcription blocks. XPB and XPD proteins are components of transcription factor TFIIH, which is involved in both basal and activated transcription. XPB is part of the core of TFIIH and has a central role in transcription, whereas XPD connects the core to the CAK subcomplex, and can tolerate many different mutations. Subtle differences in the effects of these different mutations on the many activities of TFIIH and on its stability determine the clinical outcomes, which can be XP, TTD, XP with CS, XP with TTD or COFS. Features of single and double mutant mice indicate that the neurological and ageing features associated with these disorders result from the defects in NER in association with the transcriptional deficiencies. Skin tumours in XP patients have mutations characteristic of UV-induction in the ras, p53 and ptch genes, showing that sunlight-induced mutations in these genes are important in carcinogenesis in XP patients.

Aging↗

PubMed2002

Replication of damaged DNA in mammalian cells: new solutions to an old problem.

All cells need not only to remove damage from their DNA, but also to be able to replicate DNA containing unrepaired damage. In mammalian cells, the major process by which cells are able to replicate damaged templates is translesion synthesis, the direct synthesis of DNA past altered bases. Crucial to this process is a series of recently discovered DNA polymerases. Most of them belong to a new family of polymerases designated the Y-family, which have conserved sequences in the catalytic N-terminal half of the proteins. These polymerases have different efficiencies and specificities in vitro depending on the type of damage in the template.One of them, DNA polymerase eta, is defective in xeroderma pigmentosum variants, and overwhelming evidence suggests that this is the polymerase that carries out translesion synthesis past UV-induced cyclobutane pyrimidine dimers in vivo. DNA polymerase eta is localised in replication factories during DNA replication and accumulates at sites of stalled replication forks. Many studies have been carried out on the properties of the other polymerases in vitro, but there is as yet very little evidence for their specific roles in vivo.

Cells, Cultured↗

PubMed2002

Localization of DNA polymerases eta and iota to the replication machinery is tightly co-ordinated in human cells.

Active Transport, Cell Nucleus↗

PubMed2002

Reduced level of the repair/transcription factor TFIIH in trichothiodystrophy.

Trichothiodystrophy (TTD) is a rare hereditary multisystem disorder associated with defects in nucleotide excision repair (NER) as a consequence of mutations in XPD, XPB or TTDA, three genes that are all related to TFIIH, the multiprotein complex involved in NER and transcription. Here we show that all the mutations found in TTD cases, irrespective of whether they are homozygotes, hemizygotes or compound heterozygotes, cause a substantial and specific reduction (by up to 70%) in the cellular concentration of TFIIH. Intriguingly, the degree of reduction in the level of TFIIH does not correlate with the severity of the pathological phenotype, suggesting that the severity of the clinical features in TTD cannot be related solely to the effects of mutations on the stability of TFIIH. We have also measured TFIIH levels in cells in which different mutations in the XPD gene are associated with clinical symptoms not of TTD but of the highly cancer-prone disorder xeroderma pigmentosum (XP). We have found mild reductions (up to 40%) in TFIIH content in some but not all of these cell strains. We conclude that the severity of the clinical features in TTD patients and the clinical outcome of differentially mutated XPD proteins is likely to depend both on the effects that each mutation has on the stability of TFIIH and on the transcriptional activity of the residual TFIIH complexes.

Abnormalities, Multiple↗

PubMed2002

Molecular analysis of mutations in DNA polymerase eta in xeroderma pigmentosum-variant patients.

Xeroderma pigmentosum variant (XP-V) cells are deficient in their ability to synthesize intact daughter DNA strands after UV irradiation. This deficiency results from mutations in the gene encoding DNA polymerase eta, which is required for effecting translesion synthesis (TLS) past UV photoproducts. We have developed a simple cellular procedure to identify XP-V cell strains, and have subsequently analyzed the mutations in 21 patients with XP-V. The 16 mutations that we have identified fall into three categories. Many of them result in severe truncations of the protein and are effectively null alleles. However, we have also identified five missense mutations located in the conserved catalytic domain of the protein. Extracts of cells falling into these two categories are defective in the ability to carry out TLS past sites of DNA damage. Three mutations cause truncations at the C terminus such that the catalytic domains are intact, and extracts from these cells are able to carry out TLS. From our previous work, however, we anticipate that protein in these cells will not be localized in the nucleus nor will it be relocalized into replication foci during DNA replication. The spectrum of both missense and truncating mutations is markedly skewed toward the N-terminal half of the protein. Two of the missense mutations are predicted to affect the interaction with DNA, the others are likely to disrupt the three-dimensional structure of the protein. There is a wide variability in clinical features among patients, which is not obviously related to the site or type of mutation.

Cell Line↗

PubMedDate not supplied

Replication of damaged DNA.

DNA damage is generated continually inside cells. In order to be able to replicate past damaged bases (translesion synthesis), the cell employs a series of specialised DNA polymerases, which singly or in combination, are able to bypass many different types of damage. The polymerases have similar structural domains to classical polymerases, but they have a more open structure to allow altered bases to fit into their active sites. Although not required for replication of undamaged DNA, some at least of these polymerases are located in replication factories. Emerging evidence suggests that the polymerase switch from replicative to translesion polymerases might be mediated by post-translational modifications.

Animals↗