TY - JOUR
T1 - Stepwise requirements for polymerases δ and θ in theta-mediated end joining
AU - Stroik, Susanna
AU - Carvajal-Garcia, Juan
AU - Gupta, Dipika
AU - Edwards, Alyssa
AU - Luthman, Adam
AU - Wyatt, David W.
AU - Dannenberg, Rachel L.
AU - Feng, Wanjuan
AU - Kunkel, Thomas A.
AU - Gupta, Gaorav P.
AU - Hedglin, Mark
AU - Wood, Richard
AU - Doublié, Sylvie
AU - Rothenberg, Eli
AU - Ramsden, Dale A.
N1 - Publisher Copyright:
© 2023, The Author(s), under exclusive licence to Springer Nature Limited.
PY - 2023/11/23
Y1 - 2023/11/23
N2 - Timely repair of chromosomal double-strand breaks is required for genome integrity and cellular viability. The polymerase theta-mediated end joining pathway has an important role in resolving these breaks and is essential in cancers defective in other DNA repair pathways, thus making it an emerging therapeutic target 1. It requires annealing of 2–6 nucleotides of complementary sequence, microhomologies, that are adjacent to the broken ends, followed by initiation of end-bridging DNA synthesis by polymerase θ. However, the other pathway steps remain inadequately defined, and the enzymes required for them are unknown. Here we demonstrate requirements for exonucleolytic digestion of unpaired 3′ tails before polymerase θ can initiate synthesis, then a switch to a more accurate, processive and strand-displacing polymerase to complete repair. We show the replicative polymerase, polymerase δ, is required for both steps; its 3′ to 5′ exonuclease activity for flap trimming, then its polymerase activity for extension and completion of repair. The enzymatic steps that are essential and specific to this pathway are mediated by two separate, sequential engagements of the two polymerases. The requisite coupling of these steps together is likely to be facilitated by physical association of the two polymerases. This pairing of polymerase δ with a polymerase capable of end-bridging synthesis, polymerase θ, may help to explain why the normally high-fidelity polymerase δ participates in genome destabilizing processes such as mitotic DNA synthesis 2 and microhomology-mediated break-induced replication 3.
AB - Timely repair of chromosomal double-strand breaks is required for genome integrity and cellular viability. The polymerase theta-mediated end joining pathway has an important role in resolving these breaks and is essential in cancers defective in other DNA repair pathways, thus making it an emerging therapeutic target 1. It requires annealing of 2–6 nucleotides of complementary sequence, microhomologies, that are adjacent to the broken ends, followed by initiation of end-bridging DNA synthesis by polymerase θ. However, the other pathway steps remain inadequately defined, and the enzymes required for them are unknown. Here we demonstrate requirements for exonucleolytic digestion of unpaired 3′ tails before polymerase θ can initiate synthesis, then a switch to a more accurate, processive and strand-displacing polymerase to complete repair. We show the replicative polymerase, polymerase δ, is required for both steps; its 3′ to 5′ exonuclease activity for flap trimming, then its polymerase activity for extension and completion of repair. The enzymatic steps that are essential and specific to this pathway are mediated by two separate, sequential engagements of the two polymerases. The requisite coupling of these steps together is likely to be facilitated by physical association of the two polymerases. This pairing of polymerase δ with a polymerase capable of end-bridging synthesis, polymerase θ, may help to explain why the normally high-fidelity polymerase δ participates in genome destabilizing processes such as mitotic DNA synthesis 2 and microhomology-mediated break-induced replication 3.
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U2 - 10.1038/s41586-023-06729-7
DO - 10.1038/s41586-023-06729-7
M3 - Article
C2 - 37968395
AN - SCOPUS:85176437927
SN - 0028-0836
VL - 623
SP - 836
EP - 841
JO - Nature
JF - Nature
IS - 7988
ER -