RESUMEN
The majority of STR loci are not ideal for the analysis of forensic samples with degraded and/or low template DNA. One alternative to overcome these limitations is the use of bi-allelic markers, which have low mutation rates and shorter amplicons. Human identification (HID) InDel marker panels have been described in several countries, including Brazil. The commercial kit available is, however, mostly suitable for Europeans, with lower discrimination power for other population groups. Recently, a combination of 49 InDel markers used in four different ethnic groups in the USA has been shown to be more informative than another panel from Portugal, already tested in a Rio de Janeiro sample. However, these 49 InDels have yet to be applied to other admixed or isolated populations. We assessed the efficiency of this panel in two urban admixed populations (Rio de Janeiro, Brazil; Tripoli, Libya) and one isolated Native Brazilian community. All markers are in Hardy-Weinberg equilibrium (HWE) after the Bonferroni correction, and no Linkage disequilibrium was detected. Assuming loci independence and no substructure effect, cumulative RMP was 2.7×10(-18), 1.5×10(-20), and 4.5×10(-20) for Native Brazilian, Rio de Janeiro, and Tripoli populations, respectively. The overall Fst value was 0.05512. Rio de Janeiro and Tripoli showed similar admixture levels, however for Native Brazilians one parental cluster represented over 60 % of the total parental population. We conclude that this panel is suitable for HID on these urban populations, but is less efficient for the isolated group.
Asunto(s)
Etnicidad/genética , Mutación INDEL , Brasil , Frecuencia de los Genes , Marcadores Genéticos , Humanos , Indígenas Sudamericanos/genética , Libia , Población UrbanaRESUMEN
The PriA protein of Escherichia coli provides a vital link between recombination and DNA replication. To establish the molecular basis for this link, we investigated the ability of PriA to target DNA substrates modelled on D-loops, the intermediates formed during the early stages of RecA-mediated recombination. We show that PriA binds D-loops and unwinds the DNA in reactions that rely on its ability to function as a helicase. The minimal structure that binds PriA is a duplex DNA molecule with unpaired single strands at one end, an arrangement likely to occur at a D-loop. It resembles features of the stem-loop formed by primosome assembly site (PAS) sequences in the DNA of bacteriophage phiX174 and plasmid ColE1, and which enable PriA to assemble active primosomes for the initiation of lagging strand synthesis. We suggest that PAS sequences may have evolved to mimic the natural D-loop target for PriA formed in the chromosome of E. coli during recombination and DNA repair. Genetic studies have revealed an interaction between PriA and RecG, a DNA helicase that drives branch migration of recombination intermediates. We therefore compared PriA and RecG for their ability to bind and unwind DNA. RecG, like PriA, binds D-loops and unwinds the DNA. However, it prefers branched structures with at least two duplex components. The possibility that it competes with PriA for binding recombination intermediates is discussed.
Asunto(s)
Adenosina Trifosfatasas/genética , Proteínas Bacterianas/genética , ADN Helicasas/genética , Replicación del ADN , Proteínas de Escherichia coli , Escherichia coli/genética , Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/enzimología , Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/metabolismo , Relación Estructura-ActividadRESUMEN
The RecG protein of Escherichia coli is a structure-specific DNA helicase that targets strand exchange intermediates in genetic recombination and drives their branch migration along the DNA. Strains carrying null mutations in recG show reduced recombination and DNA repair. Suppressors of this phenotype, called srgA, were located close to metB and shown to be alleles of priA. Suppression depends on the RecA, RecBCD, RecF, RuvAB, and RuvC recombination proteins. Nine srgA mutations were sequenced and shown to specify mutant PriA proteins with single amino acid substitutions located in or close to one of the conserved helicase motifs. The mutant proteins retain the ability to catalyze primosome assembly, as judged by the viability of recG srgA and srgA strains and their ability to support replication of plasmids based on the ColE1 replicon. Multicopy priA+ plasmids increase substantially the recombination- and repair-deficient phenotype of recG strains and confer similar phenotypes on recG srgA double mutants but not on ruvAB or wild-type strains. The multicopy effect is eliminated by K230R, C446G, and C477G substitutions in PriA. It is concluded that the 3'-5' DNA helicase/translocase activity of PriA inhibits recombination and that this effect is normally countered by RecG.