IN-FRAME RECOMBINATION BETWEEN THE YEAST H-ATPASE ISOGENES PMA1 AND PMA2 - INSIGHTS INTO THE MECHANISM OF RECOMBINATION INITIATED BY A DOUBLE-STRAND BREAK()

Chimeric PMA1::PMA2 sequences, placed under the control of the PMA1 pr omoter, were constructed by in vivo recombination between a gapped lin earized plasmid containing the PMA2 gene and four different fragments of the PMA1 gene. Correct in-frame assembly of the PMA sequences was s creened by the expression of the lacZ reporter gene fused to the PMA2 coding region, Restriction and sequencing analysis of 35 chimeras show ed that in all cases, the hybrid sequences were obtained as fusions be tween continuous sequences specific to PMA1 and PMA2, separated by a r egion of identity, In all but three cases, the junction sequences were not located at regions of greatest identity, Strikingly, depending on the PMA1 fragment used, junction distribution fell into two categorie s, In the first, the junctions were scattered over several hundreds of nucleotides upstream of the extremity of the PMA1 fragment, while in the second, they were concentrated at this extremity, Analysis of the alignment of the PMA1 and PMA2 sequences suggests that the distributio n is not related to the size of the region of identity at the PMA1-PMA 2 boundary but depends on the degree of identity of the PMA genes upst ream of the region of identity, the accumulation of successive mismatc hes leading to a clustered distribution of the junctions, Moreover, th e introduction of seven closely spaced mismatches near the end of a PM A1 segment with an otherwise-high level of identity with PMA2 led to a significantly increased concentration of the junctions near this end, These data show that a low level of identity in the vicinity of the c ommon boundary stretch is a strong barrier to recombination, In contra st, consecutive mismatches or regions of overall moderate identity whi ch are located several hundreds of nucleotides upstream from the PMA1 end do not necessarily block recombination.