Figure 7

Possible mtDNA fork rescue mechanisms as interpreted from existing data. (A) Pol γ stalls at a lesion (red triangle) on the leading strand template. In cultured cells, stalling of the leading strand (marked in blue) results in accumulation of partially single-stranded (ss)DNA replication intermediates, increasing the abundance of the smy-forms seen as in the UV- treated control cells. (B) Stalling of the leading strand enables the lagging strand synthesis (pink) to catch up with the replication fork, resulting also in increase of fully double-stranded, S1 nuclease resistent replication forks (y-forms). (C) TWNK continues unwinding and PrimPol re-primes the leading strand synthesis downstream of the lesion²². PrimPol also frequently primes the lagging strand synthesis, contributing to the fully dsDNA replication intermediates. Consequently, the accumulation of dsDNA y-forms at the expense of smy is especially pronounced when TWNK helicase is stalled (Supplementary Fig. S3), as also the lagging strand synthesis is halted. (D) Replication initiation from unconventional origins could initiate the rescue of the stalled forks by convergence, giving rise to replication bubbles (b in B, Fig. 6) outside of the canonical origins. (E) If the leading H-strand replication remains stalled for longer periods, L-strand replication can bypass it. In other systems, fork regression is initiated by peeling back the lagging strand (i–ii), enabling the filling in of the stalled leading strand using the nascent lagging strand as a template and resulting in fork stabilization to a chicken-foot structure (ii). If the blocking DNA lesion is repaired, the regressed part of the fork is cleaved and the fork re-started from the leading strand 3′-end (iii). Alternatively, recession of the 5′-end of the filled-in fork will generate a free 3′-tail that can be used for homology-driven strand-invasion upstream of the lesion (iv), eventually resulting in Holliday-junction formation once replication is completed (v). (F) In cultured cells broken forks are mostly degraded in an MGME1-dependent manner. However, double-strand breaks can also be repaired by homology-dependent replication after 5′-end recession. The 3′-overhang at each end of the broken molecule will strand-invade an intact template and replicate the missing piece (break-induced replication)²³. A Holliday-junction (x-form) will form once the replication is completed. Cleavage of Holliday-junctions can generate both monomeric as well as dimeric molecules (Figs 3, 5 and S4).