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Biomedical subjects

James P J Chong

Publications and source records attributed to James P J Chong.

5 recordsLinked to original sources
PubMed2006

The Methanothermobacter thermautotrophicus ExoIII homologue Mth212 is a DNA uridine endonuclease.

The genome of Methanothermobacter thermautotrophicus, as a hitherto unique case, is apparently devoid of genes coding for general uracil DNA glycosylases, the universal mediators of base excision repair following hydrolytic deamination of DNA cytosine residues. We have now identified protein Mth212, a member of the ExoIII family of nucleases, as a possible initiator of DNA uracil repair in this organism. This enzyme, in addition to bearing all the enzymological hallmarks of an ExoIII homologue, is a DNA uridine endonuclease (U-endo) that nicks double-stranded DNA at the 5'-side of a 2'-d-uridine residue, irrespective of the nature of the opposing nucleotide. This type of activity has not been described before; it is absent from the ExoIII homologues of Escherichia coli, Homo sapiens and Methanosarcina mazei, all of which are equipped with uracil DNA repair glycosylases. The U-endo activity of Mth212 is served by the same catalytic center as its AP-endo activity.

Amino Acid Sequence↗
PubMed2006

Minichromosome maintenance helicase activity is controlled by N- and C-terminal motifs and requires the ATPase domain helix-2 insert.

The minichromosome maintenance (MCM) proteins are essential conserved proteins required for DNA replication in archaea and eukaryotes. MCM proteins are believed to provide the replicative helicase activity that unwinds template DNA ahead of the replication fork. Consistent with this hypothesis, MCM proteins can form hexameric complexes that possess ATP-dependent DNA unwinding activity. The molecular mechanism by which the energy of ATP hydrolysis is harnessed to DNA unwinding is unknown, although the ATPase activity has been attributed to a highly conserved AAA+ family ATPase domain. Here we show that changes to N- and C-terminal motifs in the single MCM protein from the archaeon Methanothermobacter thermautotrophicus (MthMCM) can modulate ATP hydrolysis, DNA binding, and duplex unwinding. Furthermore, these motifs appear to influence the movement of the beta-alpha-beta insert in helix-2 of the MCM ATPase domain. Removal of this motif from MthMCM increased dsDNA-stimulated ATP hydrolysis and increased the affinity of the mutant complex for ssDNA and dsDNA. Deletion of the helix-2 insert additionally resulted in the abrogation of DNA unwinding. Our results provide significant insight into the molecular mechanisms used by the MCM helicase to both regulate and execute DNA unwinding.

Adenosine Triphosphatases↗
PubMed2005

DNA content and nucleoid distribution in Methanothermobacter thermautotrophicus.

Flow cytometry and epifluorescence microscopy results for the euryarchaeon Methanothermobacter thermautotrophicus were consistent with filaments containing multiple cells. Filaments of one to four cells contained two to eight nucleoids. Single chromosome-containing cells were not observed. Filaments containing multiple genome copies displayed synchronous DNA replication initiation. Chromosome segregation occurred during replication or rapidly after replication termination.

Cell Cycle↗
PubMed2004

A novel nuclease-ATPase (Nar71) from archaea is part of a proposed thermophilic DNA repair system.

We have identified a novel structure-specific nuclease in highly fractionated extracts of the thermophilic archaeon Methanothermobacter thermautotrophicus (Mth). The 71 kDa protein product of open reading frame mth1090 is a nuclease with ATPase activity, which we call Nar71 (Nuclease-ATPase in Repair, 71 kDa). The nar71 gene is located in a gene neighbourhood proposed by genomics to encode a novel DNA repair system conserved in thermophiles. The biochemical characterization of Nar71 presented here is the first analysis from within this neighbourhood, and it supports the insight from genomics. Nuclease activity of Nar71 is specific for 3' flaps and flayed duplexes, targeting single-stranded DNA (ssDNA) regions. This activity requires Mg2+ or Mn2+ and is greatly reduced in ATP. In ATP, Nar71 displaces ssDNA, also with high specificity for 3' flap and flayed duplex DNA. Strand displacement is weak compared with nuclease activity, but in ATPS it is abolished, suggesting that Nar71 couples ATP hydrolysis to DNA strand separation. ATPase assays confirmed that Nar71 is stimulated by ssDNA, though not double-stranded DNA. Mutation of Lys-117 in Nar71 abolished ATPase and nuclease activity, and we describe a separation-of-function mutant (K68A) that has lost ATPase activity but retains nuclease activity. A model of possible Nar71 function in DNA repair is presented.

Adenosine Triphosphatases↗