‘DNA damaging agents’ cause damage to numerous cellular molecules and do not only damage DNA. In response to treatment with these agents, cells modulate the expression levels of genes in several different pathways. The results of this study show that many proteins and pathways are needed for recovery from specific types of damage. Previously, results from genome-wide RNAi screens have suggested the involvement of a vast repertoire of DNA damage recovery proteins. Among these are proteins involved in mRNA processing, chromatin binding and Charcot Marie Tooth-disease 
. Here, as an alternative to genome-wide RNAi screens, we have implemented a focused approach where we take advantage of previous results from model organisms, such as S. cerevisiae
, and extensive knowledge of protein-protein interactions and interactomes.
Among the human homologs of toxicity-modulating yeast proteins, 25 of the 34 proteins were found to be toxicity-modulating in human cells. The three DNA damaging agents, MMS, 4-NQO and t-BuOOH, revealed distinct toxicity profiles with proteins specifically conferring resistance or sensitivity to at least one of the three agents. Deficiency for only one of the proteins, the largely uncharacterized suppressor of actin mutations 1-like SACM1L 
, modulated toxicity for all three damaging agents. SACM1L is a phosphatase, regulating Golgi morphology 
; interestingly, deletion of the yeast homolog of this protein caused cellular sensitivity to all four damaging agents tested in the yeast screen 
One striking result of our study is that we have identified many proteins that, when their mRNA levels are reduced, cause relative resistance to the DNA damaging agents, even though the proteins were selected based on the corresponding yeast deletion strains being sensitive to the agents. This observation was particularly noteworthy after exposure to 4-NQO for the members of histone modifier complexes, RNF20, SIN3A, CDC40, topoisomerase TOP3A and RNA polymerase subunit POLR2D. Possibly, this is a reflection of differential maintenance of chromatin structure in mammals versus S. cerevisiae
. While the discrepancy is puzzling, it has been shown previously that even within the same organism, cells of different origin can display distinctive, even opposite, phenotypes after being exposed to damaging agents 
. One dramatic example is that while mouse ES cells deficient in the Aag glycosylase are MMS sensitive, relative to WT, myeloid bone marrow cells and retinal rods and cones deficient in the same enzyme are extremely MMS resistant 
Vesicle transporters were among the unexpected toxicity-modulators in the yeast gene deletion screen. The endosomal vesicle transport, especially the late endosomal/lysosomal transport, is used for degradation of biomaterial, a process that intersects with the pathway of autophagy. This group of proteins is highly conserved between yeast and humans. Interestingly, the classical autophagy proteins (ATG1-ATG31) were not overrepresented among the sensitive yeast deletion strains, suggesting an alternative autophagy-like path taken after treatment with DNA damaging agents. Processing by autophagosomes/lysosomes and proteasome are two ways to clear the cell of proteins and other biomaterial. The autophagosomes can be generated from the cytoplasm but can also be derived from the trans-Golgi, when cells are exposed to the topoisomerase II inhibitor etoposide 
. The previously described role of autophagy in DNA damage response has usually been linked to the cellular death program, as several genotoxic agents have been shown to induce autophagic cell death 
. In contrast, this study suggests that cells escape cell death by induction of autophagy, because when autophagy is reduced, cells are more sensitive to MMS. This is supported by other recent studies that show that damage can also lead to non-lethal autophagy 
. The study of autophagy is also important from a clinical viewpoint because autophagy has been shown to suppress tumorigenesis 
, as well as clearing cells that contain protein aggregates such as those formed in Huntinton's disease 
Here, we have confirmed that telomere-specific proteins are needed for cells to recover after treatment DNA damaging agents. Telomere proteins in yeast are also involved in DNA damage repair, but here we have shown that specific loss of telomere maintenance, by reducing the protein levels of members of the shelterin complex, results in sensitivity to alkylating damage. We have also identified new toxicity-modulating proteins involved in chromatin modification. Previously, it was known that another Ada2-homolog, TADA2B a mammalian paralog of TADA2A, is needed in the cellular response to UV irradiation. This adaptor protein is part of the STAGA (homologous to SAGA in yeast) histone acetylation complex and is required for transcription of p53 responsive elements after UV 
. TADA2A on the other hand is a component of the similar histone acetylation complex PCAF, whose activity was recently implicated in the p53 pathway 
. The Ada2 homologs have also been found in H2B deubiquitination complexes. Ubiquitination of H2B is performed by the ubiquitin ligase RNF20, also identified as a toxicity-modulator in this study. Interestingly, a component involved in the deubiquitination of H2B (TADAD2A) has the reverse toxicity-modulation compared to an H2B ubiquitin ligase (RNF20). Other studies have shown that depletion of RNF20 inhibits both G1
arrest and apoptosis, but stimulates tumor advancement; its promotor is often hypermethylated in tumors 
. Further, RNF20 ser-522 has been identified as an ATM/ATR phosphorylation substrate after exposure to ionizing radiation 
. Depletion of the human RNF20 paralog RNF40 was recently shown to stimulate cell growth and cell migration 
. RNF20/RNF40-mediated ubiquitination of H2B is a prerequisite for RNA PolII transcription, possibly explaining the observed similarity in toxicity-modulation between RNF20 and RNA PolII subunit D (POLR2D) ().
Based on yeast orthology and conserved network structures, we have identified several human proteins necessary for recovery after cellular damage, among them components of autophagy and chromatin modifiers. Clearly, the functional relationships between yeast and human homologs are complex as the lack of some proteins conferred sensitivity in yeast cells but in human cells resulted in resistance as compared to their WT counterparts. Nevertheless, by studying the machinery that surrounds the core DNA repair proteins, we obtain a better understanding of the way cells respond to genotoxic insults. Most of the identified toxicity-modulating proteins have not been linked to DNA repair, cell cycle arrest or cell death and highlight the vast array of proteins that are involved in damage recovery after exposure to DNA damaging agents.