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Alternative lengthening of telomeres (ALT) is a telomere-elongation mechanism observed in ∼15% of cancer subtypes. Current models indicate that ALT is mediated by homology-directed repair mechanisms. By disrupting MSH6 gene expression, we show that the deficiency of MutSα (MSH2/MSH6) DNA mismatch repair complex causes striking telomere hyperextension. Mechanistically, we show MutSα is specifically recruited to telomeres in ALT cells by associating with the proliferating-cell nuclear antigen (PCNA) subunit of the ALT telomere replisome. We also provide evidence that MutSα counteracts Bloom (BLM) helicase, which adopts a crucial role in stabilizing hyper-extended telomeres and maintaining the survival of MutSα-deficient ALT cancer cells. Lastly, we propose a model in which MutSα deficiency impairs heteroduplex rejection, leading to premature initiation of telomere DNA synthesis that coincides with an accumulation of telomere variant repeats (TVRs). These findings provide evidence that the MutSα DNA mismatch repair complex acts to restrain unwarranted ALT. [Display omitted] • Loss of MutSα mismatch repair complex causes telomere hyper-extension by ALT pathway • MutSα counteracts BLM and limits premature initiation of telomere extension • Simultaneous loss of MutSα and BLM leads to cell death Barroso-Gonzalez et al. show that the mismatch repair complex MutSα restricts the alternative lengthening of telomeres (ALT) pathway in cancer cells. MutSα has an anti-recombination function and limits recombination between heteroduplex sequences at telomeres, in part by counteracting the Bloom helicase (BLM). [ABSTRACT FROM AUTHOR]

Alternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in aggressive cancers. We show that the disruption of RAD51-associated protein 1 (RAD51AP1) in ALT+ cancer cells leads to generational telomere shortening. This is due to RAD51AP1's involvement in RAD51-dependent homologous recombination (HR) and RAD52-POLD3-dependent break induced DNA synthesis. RAD51AP1 KO ALT+ cells exhibit telomere dysfunction and cytosolic telomeric DNA fragments that are sensed by cGAS. Intriguingly, they activate ULK1-ATG7-dependent autophagy as a survival mechanism to mitigate DNA damage and apoptosis. Importantly, RAD51AP1 protein levels are elevated in ALT+ cells due to MMS21 associated SUMOylation. Mutation of a single SUMO-targeted lysine residue perturbs telomere dynamics. These findings indicate that RAD51AP1 is an essential mediator of the ALT mechanism and is co-opted by post-translational mechanisms to maintain telomere length and ensure proliferation of ALT+ cancer cells. • Disruption of RAD51AP1 inhibits telomere homology-directed repair in ALT cancer cells • cGAS-ULK1-ATG7-dependent autophagy is activated upon telomere dysfunction • RAD51AP1 is specifically stabilized and regulated by SUMOylation Barroso-González et al. identify that disruption of the HR accessory factor RAD51AP1 disrupts ALT telomere elongation, causing telomere damage and fragmentation. Rather than dying, these cancer cells activate autophagy as a survival mechanism. The importance of RAD51AP1 in ALT cancer cells is underscored by its specific stabilization by SUMOylated mediated mechanisms. [ABSTRACT FROM AUTHOR]

Alternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in subsets of aggressive cancer. Recent studies have revealed that telomere repeat-containing RNA (TERRA) promotes ALT-associated HDR (ALT-HDR). Here, we report that RAD51AP1, a crucial ALT factor, interacts with TERRA and utilizes it to generate D- and R-loop HR intermediates. We also show that RAD51AP1 binds to and might stabilize TERRA-containing R-loops as RAD51AP1 depletion reduces R-loop formation at telomere DNA breaks. Proteomic analyses uncover a role for RAD51AP1-mediated TERRA R-loop homeostasis in a mechanism of chromatin-directed suppression of TERRA and prevention of transcription-replication collisions (TRCs) during ALT-HDR. Intriguingly, we find that both TERRA binding and this non-canonical function of RAD51AP1 require its intrinsic SUMO-SIM regulatory axis. These findings provide insights into the multi-contextual functions of RAD51AP1 within the ALT mechanism and regulation of TERRA. [Display omitted] • RAD51AP1 and TERRA interact and stimulate the formation of D and R loops • TERRA R-loops generated by RAD51AP1 regulate repressive chromatin at telomeres • RAD51AP1 protects telomeric regions from transcription-replication collisions Kaminski et al. show the cooperative roles of RAD51AP1 and TERRA in generating HR intermediates during ALT-HDR. Proteomic analyses uncover that RAD51AP1 binding of R-loops might serve to maintain chromatin that suppresses TERRA and prevents transcription-replication collisions (TRCs) during ALT-HDR. [ABSTRACT FROM AUTHOR]