Metnase Mediates Loading of Exonuclease 1 onto Single Strand Overhang DNA for End Resection at Stalled Replication Forks

Hyun-Suk Kim,Elizabeth A Williamson,Jac A Nickoloff,Robert A Hromas,Suk-Hee Lee

doi:10.1074/jbc.m116.745646

Hyun-Suk Kim, Elizabeth A Williamson + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m116.745646

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Abstract

Stalling at DNA replication forks generates stretches of single-stranded (ss) DNA on both strands that are exposed to nucleolytic degradation, potentially compromising genome stability. One enzyme crucial for DNA replication fork repair and restart of stalled forks in human is Metnase (also known as SETMAR), a chimeric fusion protein consisting of a su(var)3-9, enhancer-of-zeste and trithorax (SET) histone methylase and transposase nuclease domain. We previously showed that Metnase possesses a unique fork cleavage activity necessary for its function in replication restart and that its SET domain is essential for recovery from hydroxyurea-induced DNA damage. However, its exact role in replication restart is unclear. In this study, we show that Metnase associates with exonuclease 1 (Exo1), a 5'-exonuclease crucial for 5'-end resection to mediate DNA processing at stalled forks. Metnase DNA cleavage activity was not required for Exo1 5'-exonuclease activity on the lagging strand daughter DNA, but its DNA binding activity mediated loading of Exo1 onto ssDNA overhangs. Metnase-induced enhancement of Exo1-mediated DNA strand resection required the presence of these overhangs but did not require Metnase's DNA cleavage activity. These results suggest that Metnase enhances Exo1-mediated exonuclease activity on the lagging strand DNA by facilitating Exo1 loading onto a single strand gap at the stalled replication fork.

Highlights

Stalling at DNA replication forks generates stretches of single-stranded DNA on both strands that are exposed to nucleolytic degradation, potentially compromising genome stability
These results suggest that Metnase enhances exonuclease 1 (Exo1)-mediated exonuclease activity on the lagging strand DNA by facilitating Exo1 loading onto a single strand gap at the stalled replication fork
Repair pathway choice at stalled forks is important for genomic stability because unopposed classical non-homologous end joining, such as seen in malignancies with inherited BRCA1 or BRCA2 deficiencies, results in fusion of the one-sided DNA ends at damaged replication forks (12, 18 –22)

Summary

Edited by Patrick Sung

Stalling at DNA replication forks generates stretches of single-stranded (ss) DNA on both strands that are exposed to nucleolytic degradation, potentially compromising genome stability. Metnase-induced enhancement of Exo1-mediated DNA strand resection required the presence of these overhangs but did not require Metnase’s DNA cleavage activity. Repair pathway choice at stalled forks is important for genomic stability because unopposed classical non-homologous end joining (cNHEJ), such as seen in malignancies with inherited BRCA1 or BRCA2 deficiencies, results in fusion of the one-sided DNA ends at damaged replication forks (12, 18 –22). DNA resection decreases cNHEJ efficiency [27] but is essential for the homology-mediated DSB repair pathways, alternative end joining (alt-EJ), and HR (17, 29 –31). Metnase mediates loading of Exo onto single strand overhangs and enhances Exo1-mediated resection, suggesting that Metnase enhances Exo1’s 5Ј-exonuclease activity on the lagging daughter DNA by mediating Exo loading onto gapped DNA

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Experimental Procedures