When DNA replication stalls at a fork-blocking lesion, cells use damage tolerance pathways to continue replication. One pathway, ''translesion synthesis,'' involves specialized DNA polymerases that can use damaged DNA as a template. Translesion synthesis can result in mutations (i.e., can be error-prone), but it can also be error-free. An alternative pathway has been hypothesized (sometimes called ''damage avoidance''), by which cells make temporary use of an undamaged copy of the blocked sequence as a template, i.e., the newly synthesized daughter strand of the sister duplex or the allelic copy. This pathway is error-free. Evidence of the use of the daughter strand of the sister duplex as a template in intact mammalian cells has not been available heretofore. To determine whether hMms2, a ubiquitin-conjugating enzyme-like protein, plays a critical role in such damage avoidance, a human fibroblast cell strain in which both error-prone translesion synthesis and error-free damage avoidance can be detected and quantified simultaneously, and several derivative strains in which expression of hMms2 protein had been eliminated or greatly decreased, were compared for their ability to avoid translesion synthesis past UV 254nm-induced DNA photoproducts. Loss of hMms2 protein eliminated the ability of the latter strains to use an allelic copy of a target gene for damage avoidance, i.e., to produce a wild-type gene from two nonfunctional allelic copies of that gene. Molecular analysis of the wild-type gene showed that this process involves gene conversion unassociated with crossing-over. That the loss of hMms2 also eliminated use of the daughter strand of the sister duplex as a template for damage avoidance could be inferred from the fact that the frequency of mutations induced by UV in the single copy HPRT gene of the derivative strains was significantly higher than that observed in the parental strain. These data indicate that hMMS2 is essential for human cells to carry out damage avoidance by using either type of homolog, and that damage avoidance and translesion synthesis are alternative pathways for tolerating fork-blocking photoproducts. D NA is constantly exposed to damaging agents. If the damage is not removed, e.g., by excision repair, before the onset of S-phase, certain kinds of lesions can block replication by the major DNA polymerase complex (1). The damage tolerance mechanisms developed by prokaryotic and eukaryotic cells to overcome such replication blocks fall into two categories: translesion synthesis and damage avoidance. Evidence suggests that translesion synthesis is a process in which specialized, distributive DNA polymerases take over for the major DNA polymerase complex to carry out DNA replication by using the damaged DNA as a template (2-5). After distributive incorporation of nucleotides past the damage, the major DNA replication complex resumes its replication activity. Translesion synthesis can be either ''error-prone'' or ''error-free,'' depending on such factors as the type of damage, its seque...