Table 1.

Sequence analysis of the nucleotide opposite the lesion in gap repair experiments performed with donors carrying mismatches as markers for strand transfer versus template switching homologous recombination mechanisms

Nucleotide opposite the lesion	Number of isolates
	Type of mismatch in donor plasmid
	T/G1	C/A2
T	32	10
C	4	32
A	0	0
G	0	0
−1	22	25
Total isolates:	58	67
Total HR gap repair events3:	36	42
Strand Transfer HR gap repair events:	89%	76%

The assay was performed essentially as previously described ²⁰. DNA mixtures containing the gap-plasmid GP21 (with a site-specific abasic site; 100 ng), and the homologous origin-less plasmids pOFGP20T/G or pOFGP20C/A (300 ng) were used to co-transform E. coli mutS cells, defective in mismatch repair. Plasmids were isolated from kan^R colonies, and subjected to DNA sequence analysis. The table shows the identity of the base located opposite the lesion in individual clones.

The construction of gap-lesion plasmids GP21 (with a synthetic abasic site), GP20 (without a lesion) were previously described ^{20; 21; 41}. The origin-less plasmids were constructed in three steps as follow: (a) Plasmid pSKSL was digested with restriction nucleases BspE1 and BsaH1, and the resulting 3340 bp fragment was ligated to a BspE1-and BsaH1-cleaved 772 bp PCR fragment, which carried BspE1 and BsaH1 restriction sites, and the origin of replication from plasmid pSKSL. This yielded plasmid pOri2 that contained two origin of replication. (b) Plasmid pOri2 was restricted with BanI, deleting a 1021 bp fragment with the original replication origin of the plasmid. The 3092 bp fragment was then self-ligated to form plasmid pOri1. (c) Finally, the ori-less plasmid carrying the mismatch was prepared by a method similar to the preparation of the gapped plasmid, except that a duplex oligonucleotide carrying the T/G or C/A mismatches was used instead of the gapped duplex. Plasmid pOri1 was cleaved with BstX I and Bsa I and the resultant large fragment, which did not contain any origin of replication, was gel-purified, and ligated to the duplex oligonucleotide carrying the mismatch. The ligation product was gel purified again. The presence of mismatches was verified by DNA sequence analysis of the two strands, and the inability to replicate was verified by demonstrating the inability of the plasmid to transform E. coli cells to kanamycin resistance.

¹In this origin-less donor plasmid the T is located opposite the site corresponding to the lesion.

²In this origin-less donor plasmid the C is located opposite the site corresponding to the lesion.

³The total HR gap repair events do not include the −1 deletions, which are independent on the presence of a homologous plasmid (Table 1s).