DNA repair in mammalian cells: Base excision repair: the long and short of it.

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).

Characterization of novel mutations in the catalytic domain of the PCSK9 gene.

OBJECTIVES: To expand our understanding of the structure and function of proprotein convertase subtilisin/kexin type 9 (PCSK9) by studying how naturally occurring mutations in PCSK9 disrupt the function of PCSK9. DESIGN: Mutations in PCSK9 were identified by sequencing of DNA from subjects with hypo- or hypercholesterolemia. The effect of the identified mutations on the autocatalytic cleavage and secretion of PCSK9, as well as the effect on PCSK9-mediated degradation of the low density lipoprotein receptors, were determined in HepG2 or HEK293 cells transiently transfected with mutant PCSK9-containing plasmids. The findings were collated to the clinical characteristics of the subjects possessing these mutations, and the phenotypic effects were analysed in terms of available structural data for PCSK9. RESULTS: Five novel mutations in PCSK9 were identified. Mutation R215H was a gain-of-function mutation which causes hypercholesterolemia. Mutation G236S and N354I were loss-of-function mutations due to failure to exit the endoplasmic reticulum or failure to undergo autocatalytic cleavage, respectively. Mutations A245T and R272Q were most likely normal genetic variants. By comparing the number of patients with gain-of-function mutations in PCSK9 with the number of familial hypercholesterolemia heterozygotes among subjects with hypercholesterolemia, the prevalence of subjects with gain-of-function mutations in PCSK9 in Norway can be estimated to one in 15,000. CONCLUSION: This study has provided novel information about the structural requirements for the normal function of PCSK9. However, more studies are needed to determine the mechanisms by which gain-of-function mutations in PCSK9 cause hypercholesterolemia.

Computational prediction of the effects of non-synonymous single nucleotide polymorphisms in human DNA repair genes.

Non-synonymous single nucleotide polymorphisms (nsSNPs) represent common genetic variation that alters encoded amino acids in proteins. All nsSNPs may potentially affect the structure or function of expressed proteins and could therefore have an impact on complex diseases. In an effort to evaluate the phenotypic effect of all known nsSNPs in human DNA repair genes, we have characterized each polymorphism in terms of different functional properties. The properties are computed based on amino acid characteristics (e.g. residue volume change); position-specific phylogenetic information from multiple sequence alignments and from prediction programs such as SIFT (Sorting Intolerant From Tolerant) and PolyPhen (Polymorphism Phenotyping). We provide a comprehensive, updated list of all validated nsSNPs from dbSNP (public database of human single nucleotide polymorphisms at National Center for Biotechnology Information, USA) located in human DNA repair genes. The list includes repair enzymes, genes associated with response to DNA damage as well as genes implicated with genetic instability or sensitivity to DNA damaging agents. Out of a total of 152 genes involved in DNA repair, 95 had validated nsSNPs in them. The fraction of nsSNPs that had high probability of being functionally significant was predicted to be 29.6% and 30.9%, by SIFT and PolyPhen respectively. The resulting list of annotated nsSNPs is available online (http://dna.uio.no/repairSNP), and is an ongoing project that will continue assessing the function of coding SNPs in human DNA repair genes.

ParAlign: a parallel sequence alignment algorithm for rapid and sensitive database searches.

There is a need for faster and more sensitive algorithms for sequence similarity searching in view of the rapidly increasing amounts of genomic sequence data available. Parallel processing capabilities in the form of the single instruction, multiple data (SIMD) technology are now available in common microprocessors and enable a single microprocessor to perform many operations in parallel. The ParAlign algorithm has been specifically designed to take advantage of this technology. The new algorithm initially exploits parallelism to perform a very rapid computation of the exact optimal ungapped alignment score for all diagonals in the alignment matrix. Then, a novel heuristic is employed to compute an approximate score of a gapped alignment by combining the scores of several diagonals. This approximate score is used to select the most interesting database sequences for a subsequent Smith-Waterman alignment, which is also parallelised. The resulting method represents a substantial improvement compared to existing heuristics. The sensitivity and specificity of ParAlign was found to be as good as Smith-Waterman implementations when the same method for computing the statistical significance of the matches was used. In terms of speed, only the significantly less sensitive NCBI BLAST 2 program was found to outperform the new approach. Online searches are available at http://dna.uio.no/search/

Six-fold speed-up of Smith-Waterman sequence database searches using parallel processing on common microprocessors.

MOTIVATION: Sequence database searching is among the most important and challenging tasks in bioinformatics. The ultimate choice of sequence-search algorithm is that of Smith-Waterman. However, because of the computationally demanding nature of this method, heuristic programs or special-purpose hardware alternatives have been developed. Increased speed has been obtained at the cost of reduced sensitivity or very expensive hardware. RESULTS: A fast implementation of the Smith-Waterman sequence-alignment algorithm using Single-Instruction, Multiple-Data (SIMD) technology is presented. This implementation is based on the MultiMedia eXtensions (MMX) and Streaming SIMD Extensions (SSE) technology that is embedded in Intel's latest microprocessors. Similar technology exists also in other modern microprocessors. Six-fold speed-up relative to the fastest previously known Smith-Waterman implementation on the same hardware was achieved by an optimized 8-way parallel processing approach. A speed of more than 150 million cell updates per second was obtained on a single Intel Pentium III 500 MHz microprocessor. This is probably the fastest implementation of this algorithm on a single general-purpose microprocessor described to date.

Cell-cycle regulation, intracellular sorting and induced overexpression of the human NTH1 DNA glycosylase involved in removal of formamidopyrimidine residues from DNA.

Endonuclease III (Nth) of Escherichia coli is a DNA glycosylase essential for the removal of oxidised pyrimidine base residues from DNA. Several eukaryotic homologues have recently been identified and shown to have biochemical properties similar to those of Nth. However, some of the eukaryotic counterparts also appear to remove imidazole ring-opened purine residues (faPy), a property not shared by the enzymes of bacterial origin. Here, we show that the human enzyme also possesses efficient faPy DNA glycosylase activity as indicated both from studies of the purified protein and induced overexpression of the human NTH1 cDNA in HeLa cells. We constructed green fluorescent protein-tagged hNTH1 fusion proteins to study the cellular localisation of hNTH1 and found strong and exclusive sorting to the nucleus. Studies with synchronised cells showed that the expression of hNTH1 is regulated during the cell cycle with increased transcription during early and mid S-phase.

The Saccharomyces cerevisiae homologues of endonuclease III from Escherichia coli, Ntg1 and Ntg2, are both required for efficient repair of spontaneous and induced oxidative DNA damage in yeast.

Endonuclease III from Escherichia coli is the prototype of a ubiquitous DNA repair enzyme essential for the removal of oxidized pyrimidine base damage. The yeast genome project has revealed the presence of two genes in Saccharomyces cerevisiae, NTG1 and NTG2, encoding proteins with similarity to endonuclease III. Both contain the highly conserved helix-hairpin-helix motif, whereas only one (Ntg2) harbors the characteristic iron-sulfur cluster of the endonuclease III family. We have characterized these gene functions by mutant and enzyme analysis as well as by gene expression and intracellular localization studies. Targeted gene disruption of NTG1 and NTG2 produced mutants with greatly increased spontaneous and hydrogen peroxide-induced mutation frequency relative to the wild type, and the mutation response was further increased in the double mutant. Both enzymes were found to remove thymine glycol and 2, 6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (faPy) residues from DNA with high efficiency. However, on UV-irradiated DNA, saturating concentrations of Ntg2 removed only half of the cytosine photoproducts released by Ntg1. Conversely, 5-hydroxycytosine was removed efficiently only by Ntg2. The enzymes appear to have different reaction modes, as judged from much higher affinity of Ntg2 for damaged DNA and more efficient borhydride trapping of Ntg1 to abasic sites in DNA despite limited DNA binding. Northern blot and promoter fusion analysis showed that NTG1 is inducible by cell exposure to DNA-damaging agents, whereas NTG2 is constitutively expressed. Ntg2 appears to be a nuclear enzyme, whereas Ntg1 was sorted both to the nucleus and to the mitochondria. We conclude that functions of both NTG1 and NTG2 are important for removal of oxidative DNA damage in yeast.

SALSA: improved protein database searching by a new algorithm for assembly of sequence fragments into gapped alignments.

MOTIVATION: Optimal sequence alignment based on the Smith-Waterman algorithm is usually too computationally demanding to be practical for searching large sequence databases. Heuristic programs like FASTA and BLAST have been developed which run much faster, but at the expense of sensitivity. RESULTS: In an effort to approximate the sensitivity of an optimal alignment algorithm, a new algorithm has been devised for the computation of a gapped alignment of two sequences. After scanning for high-scoring words and extensions of these to form fragments of similarity, the algorithm uses dynamic programming to build an accurate alignment based on the fragments initially identified. The algorithm has been implemented in a program called SALSA and the performance has been evaluated on a set of test sequences. The sensitivity was found to be close to the Smith-Waterman algorithm, while the speed was similar to FASTA (ktup = 2). AVAILABILITY: Searches can be performed from the SALSA homepage at http://dna.uio.no/salsa/ using a wide range of databases. Source code and precompiled executables are also available. CONTACT: torbjorn.rognes@labmed.uio.no

Opposite base-dependent reactions of a human base excision repair enzyme on DNA containing 7,8-dihydro-8-oxoguanine and abasic sites.

The guanine modification 7,8-dihydro-8-oxoguanine (8-oxoG) is a potent premutagenic lesion formed spontaneously at high frequencies in the genomes of aerobic organisms. We have characterized a human DNA repair glycosylase for 8-oxoG removal, hOGH1 (human yeast OGG1 homologue), by molecular cloning and functional analysis. Expression of the human cDNA in a repair deficient mutator strain of Escherichia coli (fpg mutY) suppressed the spontaneous mutation frequency to almost normal levels. The hOGH1 enzyme was localized to the nucleus in cells transfected by constructs of hOGH1 fused to green fluorescent protein. Enzyme purification yielded a protein of 38 kDa removing both formamidopyrimidines and 8-oxoG from DNA. The enzymatic activities of hOGH1 was analysed on DNA containing single residues of 8-oxoG or abasic sites opposite each of the four normal bases in DNA. Excision of 8-oxoG opposite C was the most efficient and was followed by strand cleavage via beta-elimination. However, significant removal of 8-oxoG from mispairs (8-oxoG: T >G >A) was also demonstrated, but essentially without an associated strand cleavage reaction. Assays with abasic site DNA showed that strand cleavage was indeed dependent on the presence of C in the opposite strand, irrespective of the prior removal of an 8-oxoG residue. It thus appears that strand incisions are made only if repair completion results in correct base insertion, whereas excision from mispairs preserves strand continuity and hence allows for error-free correction by a postreplicational repair mechanism.