Summary During eukaryotic DNA interstrand cross-link (ICL) repair, cross-links are resolved (“unhooked”) by nucleolytic incisions surrounding the lesion. In vertebrates, ICL repair is triggered when replication forks collide with the lesion, leading to FANCI-FANCD2-dependent unhooking and formation of a double-strand break (DSB) intermediate. Using Xenopus egg extracts, we describe here a replication-coupled ICL repair pathway that does not require incisions or FANCI-FANCD2. Instead, the ICL is unhooked when one of the two N-glycosyl bonds forming the cross-link is cleaved by the DNA glycosylase NEIL3. Cleavage by NEIL3 is the primary unhooking mechanism for psoralen- and abasic site-ICLs. When N-glycosyl bond cleavage is prevented, unhooking occurs via FANCI-FANCD2-dependent incisions. In summary, we identify an incision-independent unhooking mechanism that avoids DSB formation and represents the preferred pathway of ICL repair in a vertebrate cell-free system.

A critical step in DNA interstrand cross-link repair is the programmed collapse of replication forks that have stalled at an ICL. This event is regulated by the Fanconi anemia pathway, which suppresses bone marrow failure and cancer. In this perspective, we focus on the structure of forks that have stalled at ICLs, how these structures might be incised by endonucleases, and how incision is regulated by the Fanconi anemia pathway.

DNA interstrand cross-links (ICLs) prevent strand separation during DNA replication and transcription and are therefore extremely cytotoxic. In metazoans, a major pathway of ICL repair is coupled to DNA replication and requires the Fanconi anemia pathway. In most current models, collision of a single DNA replication fork with an ICL is sufficient to initiate repair. In contrast, we show here that in Xenopus egg extracts, two DNA replication forks must converge on an ICL to trigger repair. When only one fork reaches the ICL, the replicative CMG helicase fails to unload from the stalled fork, and repair is blocked. Arrival of a second fork, even when substantially delayed, rescues repair. We conclude that ICL repair requires a replication-induced X-shaped DNA structure surrounding the lesion, and we speculate how this requirement helps maintain genomic stability in S phase.

Accumulated evidence suggests that M2-like polarized tumor associated macrophages (TAMs) plays an important role in cancer progression and metastasis, establishing TAMs, especially M2-like TAMs as an appealing target for therapy intervention. Here we found that metformin significantly suppressed IL-13 induced M2-like polarization of macrophages, as illustrated by reduced expression of CD206, down-regulation of M2 marker mRNAs, and inhibition of M2-like macrophages promoted migration of cancer cells and endothelial cells. Metformin triggered AMPKα1 activation in macrophage and silencing of AMPKα1 partially abrogated the inhibitory effect of metformin in IL-13 induced M2-like polarization. Administration of AICAR, another activator of AMPK, also blocked the M2-like polarization of macrophages. Metformin greatly reduced the number of metastases of Lewis lung cancer without affecting tumor growth. In tumor tissues, the percentage of M2-like macrophage was decreased and the area of pericyte-coated vessels was increased. Further, the anti-metastatic effect of metformin was abolished when the animals were treated with macrophages eliminating agent clodronate liposome. These findings suggest that metformin is able to block the M2-like polarization of macrophages partially through AMPKα1, which plays an important role in metformin inhibited metastasis of Lewis lung cancer.