Arne Klungland

Reconstitution of DNA base excision-repair with purified human proteins: interaction between DNA polymerase beta and the XRCC1 protein.

Repair of a uracil‐guanine base pair in DNA has been reconstituted with the recombinant human proteins uracil‐DNA glycosylase, apurinic/apyrimidinic endonuclease, DNA polymerase beta and DNA ligase III. The XRCC1 protein, which is known to bind DNA ligase III, is not absolutely required for the reaction but suppresses strand displacement by DNA polymerase beta, allowing for more efficient ligation after filling of a single nucleotide patch. We show that XRCC1 interacts directly with DNA polymerase beta using far Western blotting, affinity precipitation and yeast two‐hybrid analyses. In addition, a complex formed between DNA polymerase beta and a double‐stranded oligonucleotide containing an incised abasic site was supershifted by XRCC1 in a gel retardation assay. The region of interaction with DNA polymerase beta is located within residues 84–183 in the N‐terminal half of the XRCC1 protein, whereas the C‐terminal region of XRCC1 is involved in binding DNA ligase III. These data indicate that XRCC1, which has no known catalytic activity, might serve as a scaffold protein during base excision‐repair. DNA strand displacement and excessive gap filling during DNA repair were observed in cell‐free extracts of an XRCC1‐deficient mutant cell line, in agreement with the results from the reconstituted system.

Second pathway for completion of human DNA base excision‐repair: reconstitution with purified proteins and requirement for DNase IV (FEN1)

Two forms of DNA base excision‐repair (BER) have been observed: a ‘short‐patch’ BER pathway involving replacement of one nucleotide and a ‘long‐patch’ BER pathway with gap‐filling of several nucleotides. The latter mode of repair has been investigated using human cell‐free extracts or purified proteins. Correction of a regular abasic site in DNA mainly involves incorporation of a single nucleotide, whereas repair patches of two to six nucleotides in length were found after repair of a reduced or oxidized abasic site. Human AP endonuclease, DNA polymerase β and a DNA ligase (either III or I) were sufficient for the repair of a regular AP site. In contrast, the structure‐specific nuclease DNase IV (FEN1) was essential for repair of a reduced AP site, which occurred through the long‐patch BER pathway. DNase IV was required for cleavage of a reaction intermediate generated by template strand displacement during gap‐filling. XPG, a related nuclease, could not substitute for DNase IV. The long‐patch BER pathway was largely dependent on DNA polymerase β in cell extracts, but the reaction could be reconstituted with either DNA polymerase β or δ. Efficient repair of γ‐ray‐induced oxidized AP sites in plasmid DNA also required DNase IV. PCNA could promote the Pol β‐dependent long‐patch pathway by stimulation of DNase IV.

Post-translational modification of poly(ADP-ribose) polymerase induced by DNA strand breaks

There are one million molecules of poly(ADP-ribose) polymerase (PARP) in mammalian cell nuclei and the enzyme is found in most eukaryotes, with the notable exception of yeasts. In response to DNA damage caused by ionizing radiation or alkylating agents, PARP binds to strand interruptions in DNA and undergoes rapid automodification with synthesis of long branched polymers of highly negatively charged poly(ADP-ribose). DNA repair occurs after dissociation of modified PARP from DNA strand breaks. Biochemical data with enzyme-depleted extracts and studies of enzyme-deficient mice show that PARP does not participate directly in DNA repair. Possible roles for poly(ADP-ribose) synthesis are discussed.

ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility.

N(6)-methyladenosine (m(6)A) is the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes. Here we report ALKBH5 as another mammalian demethylase that oxidatively reverses m(6)A in mRNA in vitro and in vivo. This demethylation activity of ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles. Alkbh5-deficient male mice have increased m(6)A in mRNA and are characterized by impaired fertility resulting from apoptosis that affects meiotic metaphase-stage spermatocytes. In accordance with this defect, we have identified in mouse testes 1,551 differentially expressed genes that cover broad functional categories and include spermatogenesis-related mRNAs involved in the p53 functional interaction network. The discovery of this RNA demethylase strongly suggests that the reversible m(6)A modification has fundamental and broad functions in mammalian cells.

DNA Repair in Mammalian Cells

Abstract.Base excision repair (BER) is the primary DNA repair pathway that corrects base lesions that arise due to oxidative, alkylation, deamination, and depurinatiation/depyrimidination damage. BER facilitates the repair of damaged DNA via two general pathways – short-patch and long-patch. The shortpatch BER pathway leads to a repair tract of a single nucleotide. Alternatively, the long-patch BER pathway produces a repair tract of at least two nucleotides. The BER pathway is initiated by one of many DNA glycosylases, which recognize and catalyze the removal of damaged bases. The completion of the BER pathway is accomplished by the coordinated action of at least three additional enzymes. These downstream enzymes carry out strand incision, gap-filling and ligation. The high degree of BER conservation between E. coli and mammals has lead to advances in our understanding of mammalian BER. This review will provide a general overview of the mammalian BER pathway. (Part of a Multi-author Review)

OGG1 initiates age-dependent CAG trinucleotide expansion in somatic cells

Although oxidative damage has long been associated with ageing and neurological disease, mechanistic connections of oxidation to these phenotypes have remained elusive. Here we show that the age-dependent somatic mutation associated with Huntington’s disease occurs in the process of removing oxidized base lesions, and is remarkably dependent on a single base excision repair enzyme, 7,8-dihydro-8-oxoguanine-DNA glycosylase (OGG1). Both in vivo and in vitro results support a ‘toxic oxidation’ model in which OGG1 initiates an escalating oxidation–excision cycle that leads to progressive age-dependent expansion. Age-dependent CAG expansion provides a direct molecular link between oxidative damage and toxicity in post-mitotic neurons through a DNA damage response, and error-prone repair of single-strand breaks.

Base excision repair of oxidative DNA damage activated by XPG protein.

Oxidized pyrimidines in DNA are removed by a distinct base excision repair pathway initiated by the DNA glycosylase--AP lyase hNth1 in human cells. We have reconstituted this single-residue replacement pathway with recombinant proteins, including the AP endonuclease HAP1/APE, DNA polymerase beta, and DNA ligase III-XRCC1 heterodimer. With these proteins, the nucleotide excision repair enzyme XPG serves as a cofactor for the efficient function of hNth1. XPG protein promotes binding of hNth1 to damaged DNA. The stimulation of hNth1 activity is retained in XPG catalytic site mutants inactive in nucleotide excision repair. The data support the model that development of Cockayne syndrome in XP-G patients is related to inefficient excision of endogenous oxidative DNA damage.

Critical role of aquaporin-4 (AQP4) in astrocytic Ca2+ signaling events elicited by cerebral edema

Aquaporin-4 (AQP4) is a primary influx route for water during brain edema formation. Here, we provide evidence that brain swelling triggers Ca2+ signaling in astrocytes and that deletion of the Aqp4 gene markedly interferes with these events. Using in vivo two-photon imaging, we show that hypoosmotic stress (20% reduction in osmolarity) initiates astrocytic Ca2+ spikes and that deletion of Aqp4 reduces these signals. The Ca2+ signals are partly dependent on activation of P2 purinergic receptors, which was judged from the effects of appropriate antagonists applied to cortical slices. Supporting the involvement of purinergic signaling, osmotic stress was found to induce ATP release from cultured astrocytes in an AQP4-dependent manner. Our results suggest that AQP4 not only serves as an influx route for water but also is critical for initiating downstream signaling events that may affect and potentially exacerbate the pathological outcome in clinical conditions associated with brain edema.

A global DNA repair mechanism involving the Cockayne syndrome B (CSB) gene product can prevent the in vivo accumulation of endogenous oxidative DNA base damage

The Cockayne syndrome B (CSB) gene product is involved in the repair of various types of base modifications in actively transcribed DNA sequences. To investigate its significance for the repair of endogenous oxidative DNA damage, homozygous csb−/−/ogg1−/− double knockout mice were generated. These combine the deficiency of CSB with that of OGG1, a gene coding for the mammalian repair glycosylase that initiates the base excision repair of 7,8-dihydro-8-oxoguanine (8-oxoG). Compared to ogg1−/− mice, csb−/−/ogg1−/− mice were found to accumulate with age severalfold higher levels of oxidited purine modifications in hepatocytes, splenocytes and kidney cells. In contrast, the basal (steady-state) levels of oxidative DNA modifications in cells from csb−/− mice were not different from those in wild-type mice and did not increase with age. The analysis of the repair rates of additional oxidative DNA base modifications induced by photosensitization in immortalized embryonic fibroblasts was in accordance with these findings: compared to wild-type cells, the global repair was only slightly affected in csb−/− cells, more compromised in ogg1−/− cells, but virtually absent in csb−/−/ogg1−/− cells. An inhibition of transcription by α-amanitin did not block the Csb-dependent repair in ogg1−/− fibroblasts. The influence of Csb on the global repair of 8-oxoG was not detectable in assays with total protein extracts and in a shuttle vector system. The data indicate a role for Csb in the removal of 8-oxoG from the overall genome that is independent of both Ogg1-mediated base excision repair and regular transcription.

Repair deficient mice reveal mABH2 as the primary oxidative demethylase for repairing 1meA and 3meC lesions in DNA

Two human homologs of the Escherichia coli AlkB protein, denoted hABH2 and hABH3, were recently shown to directly reverse 1‐methyladenine (1meA) and 3‐methylcytosine (3meC) damages in DNA. We demonstrate that mice lacking functional mABH2 or mABH3 genes, or both, are viable and without overt phenotypes. Neither were histopathological changes observed in the gene‐targeted mice. However, in the absence of any exogenous exposure to methylating agents, mice lacking mABH2, but not mABH3 defective mice, accumulate significant levels of 1meA in the genome, suggesting the presence of a biologically relevant endogenous source of methylating agent. Furthermore, embryonal fibroblasts from mABH2‐deficient mice are unable to remove methyl methane sulfate (MMS)‐induced 1meA from genomic DNA and display increased cytotoxicity after MMS exposure. In agreement with these results, we found that in vitro repair of 1meA and 3meC in double‐stranded DNA by nuclear extracts depended primarily, if not solely, on mABH2. Our data suggest that mABH2 and mABH3 have different roles in the defense against alkylating agents.