BOD1L Is Required to Suppress Deleterious Resection of Stressed Replication Forks
Authors: Simon J Boulton
Martin R Higgs
Jadwiga Nieminuszczy
Ellis L Ryan
Wojciech Niedzwiedz
Tatjana Stankovic
Andrew N Blackford
Nicholas J Davies
John J Reynolds
Alicja Winczura
Anastasia Zlatanou
Val√©rie Borel
Edward S Miller
Grant S Stewart
Journal: Molecular Cell (2015)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Replication forks; Genome stability; Fanconi anemia; Homologous recombination; BOD1L
Affiliations: School of Cancer Sciences, University of Birmingham, Birmingham B15 2TT, UK., The Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK., The Francis Crick Institute, Clare Hall Laboratories, South Mimms, Herts EN6 3LD, UK., School of Cancer Sciences, University of Birmingham, Birmingham B15 2TT, UK. Electronic address: .

Abstract:  Recognition and repair of damaged replication forks are essential to maintain genome stability and are coordinated by the combined action of the Fanconi anemia and homologous recombination pathways. These pathways are vital to protect stalled replication forks from uncontrolled nucleolytic activity, which otherwise causes irreparable genomic damage. Here, we identify BOD1L as a component of this fork protection pathway, which safeguards genome stability after replication stress. Loss of BOD1L confers exquisite cellular sensitivity to replication stress and uncontrolled resection of damaged replication forks, due to a failure to stabilize RAD51 at these forks. Blocking DNA2-dependent resection, or downregulation of the helicases BLM and FBH1, suppresses both catastrophic fork processing and the accumulation of chromosomal damage in BOD1L-deficient cells. Thus, our work implicates BOD1L as a critical regulator of genome integrity that restrains nucleolytic degradation of damaged replication forks. 

Retracted: No

PubPeer comments:
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 Problematic data figures 5A and 6G. Legend figure 5A states  The blots originate from a single gel. A white line denotes removal of the superfluous lanes , yet in figure 5G there are no splices in the BODL1 panel. What lanes were removed if 6G is unspliced?  , 
 #2 Thank you for highlighting our mistake in relation to Figures 5A and 6G of our manuscript. We have wrongly indicated that the BOD1L Western in Figure 5A is cropped and it wasn t. Only the RPA2 Western had lanes removed. The use of the same Western blots for BOD1L, H2A and the non-specific loading control in Figures 5A and 6G is because they were run from the same extracts at the same time and individual strips were probed for different antibodies. Since Rad51 and RPA2 run at approximately the same mwt, they were run on a separate gels still using the same extracts. We ran additional extracts when we ran the RPA2 Western, which were not needed for the figure. Hence these were cropped out. Each Western strip, whether cropped or not is part of the same Western strip and is NOT an amalgamation of different Western blots. Despite our mistake, this does NOT in any way affect the result and our conclusions. , 
 #3  Only the RPA2 Western had lanes removed.  If only the RPA2 Western had lanes removed how can you align it with BOD1L, H2A and loading control in figure 5A. Does the RPA2 Western have a loading control? Could the authors post the full blots for BOD1L, RPA2 and Rad51 here? , 
 #4 The RPA2 blot can be aligned with the H2A and loading control because which we know which lanes contain the same extracts. It is not uncommon for superfluous lanes to be removed from blots as long as they indicated as such, which we have done. The H2A and loading control blots in Figure 5A are the loading controls for RPA2. The full Western strips for BOD1L, H2A and Rad51 are shown in figure 6G. We will try to dig out the original Western blots and post them. , 
 My apologies but I incorrectly said previously that the BOD1L Western in Figure 5A was not cropped. It was. Our mistake was not labeling the BOD1L Western in Figure 6G as being cropped. I have attached original scans of the blots. However, I should point out that the same extracts were used with all the Westerns with the exception that the HU treated samples were removed from some of the blots.  , 
 #6 As the BOD1L and RPA2 blots have been spliced and are from different blots than the other blots why align them with the other blots in figures 5A and 6G? , 
 #7 The BOD1L and RPA2 blots are from the same gel nitrocellulose filter that was cut into strips and blotted for the separate protein because BOD1L is 500kDa and RPA2 is 32kDa. This was achieved using a large biphasic SDS-PAGE with 6% on top and 12% on the bottom. Since Rad51 runs at the same mwt as RPA2 we could not blot for them both on the same gel. Hence we ran another Western using the same extracts, with the exception that the HU samples were not run on the second gel as we only wanted to look at MMC-induced chromatin loading of Rad51. We used the same untreated and MMC treated extracts to run both gels therefore, the levels of bound protein are directly comparable. We didn t reblot for BOD1L on the second gel we ran to assess Rad51 loading because we had already checked the level of BOD1L knockdown using these extracts and also we wanted to blot for another protein (BRCA2), which runs at roughly the same mwt (give or take) as BOD1L. We were able to blot for H2A from both gels as we can separate H2A from RPA2 and Rad51. However we only showed the H2A level for one of the gels. The level of H2A for the first gel, which was cropped, was present on the second of the three original Western blots I posted (at the bottom of the Western, run in duplicated). This also shows the level of chromatin loaded in our chromatin preps was equal. I hope this has answered your question. ,