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R-Loop in the replication origin of human mitochondrial DNA is resolved by RecG, a Holliday junction-specific helicase.

Stable RNA-DNA hybrids (R-loops) prime the initiation of replication in Escherichia coli cells. The R-loops are resolved by Escherichia coli RecG protein, a Holliday junction specific helicase. A stable RNA-DNA hybrid formation in the mitochondrial D-loop region is also implicated in priming the replication of mitochondrial DNA. Consistent with this hypothesis, the 3' ends of the mitochondrial R-loop formed by in vitro transcription are located close to the initiation sites of the mitochondrial DNA replication. This mitochondrial R-loop is resolved by RecG in a dose-dependent manner. Since the resolution by RecG requires ATP, the resolution is dependent on the helicase activity of RecG. A linear RNA-DNA heteroduplex is not resolved by RecG, suggesting that RecG specifically recognizes the higher structure of the mitochondrial R-loop. This is the first example that R-loops of an eukaryotic origin is sensitive to a junction-specific helicase. The resolution of the mitochondrial R-loop by RecG suggests that the replication-priming R-loops have a common structural feature recognized by RecG.

Bacterial Proteins↗

Characterization of a yeast replication origin (ars2) and construction of stable minichromosomes containing cloned yeast centromere DNA (CEN3).

A yeast DNA sequence (ars2), capable of supporting autonomous replication of plasmids, in yeast, has been characterized. The ars2 replicator occurs about 7 kb from the ARG4 gene on yeast chromosome VIII. Plasmids containing ars2 and the ARG4 gene transform yeast arg4 mutants to ARG4+ with high frequency (about 103 transformants/micrograms DNA) and replicate autonomously in the transformed cells. The ars2 plasmids are mitotically unstable and are readily lost from yeast cultures when grown under nonselective conditions. The addition of a DNA segment containing functional yeast centromere (CEN3) and an ars2 plasmid effectively stabilizes the plasmid against both mitotic and meiotic loss. The ars2-CEN3 minichromosomes replicate autonomously in controlled copy number while segregating in a typical Mendelian pattern (2+ :2-) during meiosis. The requirement for a separate replicator sequence for stable mitotic and meiotic maintenance of centromere-containing minichromosomes is equally satisfied by the presence of either ars1 or ars2. The centromere controls plasmid copy number to a low value (usually one) regardless of the type of replicator used.

Base Sequence↗

The distribution and properties of RNA primed initiation sites of DNA synthesis at the replication origin of Escherichia coli chromosome.

RNA-linked DNA molecules were obtained from E. coli dnaCts cells synchronously initiating a new round of chromosome replication. The deoxynucleotides at the transition from primer RNA to DNA were 32P-labeled, and their positions were located on the nucleotide sequence of 1.4 kb genomic region (position -906 to +493) including the oriC and its leftside flanking region. In the r-strand (the counterclockwise strand), many strong transition sites were mapped in the left half portion of the oriC and a few weak sites in the left outside region. In the 1-strand (the clockwise strand), no transition sites were found inside the oriC but many weak sites were found in the left outside region. The results support the initiation mechanism in which the first leading strand synthesis starts with the r-strand counterclockwise from the oriC that is followed by the 1-strand synthesis on the displaced template strand on the left of oriC. Primer RNA molecules attached to the strong r-strand transition sites were only a few residues in length. Properties of the transition sites were discussed.

Base Sequence↗

The hemimethylated replication origin of Escherichia coli can be initiated in vitro.

Unmethylated, fully methylated, and hemimethylated oriC-containing plasmids were assayed as substrates for DNA replication in vitro by using a system reconstituted with pure proteins. In contrast to the in vivo situation, all three substrates were initiated efficiently; the fully methylated plasmid was about twice as active as the other two.

Bacterial Proteins↗