AP endonuclease EXO-3 deficiency causes developmental delay and abnormal vulval organogenesis, Pvl, through DNA glycosylase-initiated checkpoint activation in Caenorhabditis elegans

Abstract

AP endonuclease deficiency causes cell death and embryonic lethality in mammals. However, the physiological roles of AP endonucleases in multicellular organisms remain unclear, especially after embryogenesis. Here, we report novel physiological roles of the AP endonuclease EXO-3 from larval to adult stages in Caenorhabditis elegans, and elucidated the mechanism of the observed phenotypes due to EXO-3 deficiency. The exo-3 mutants exhibited developmental delay, whereas the apn-1 mutants did not. The delay depended on the DNA glycosylase NTH-1 and checkpoint kinase CHK-2. The exo-3 mutants had further developmental delay when treated with AP site-generating agents such as methyl methane sulfonate and sodium bisulfite. The further delay due to sodium bisulfite was dependent on the DNA glycosylase UNG-1. The exo-3 mutants also demonstrated an increase in dut-1 (RNAi)-induced abnormal vulval organogenesis protruding vulva (Pvl), whereas the apn-1 mutants did not. The increase in Pvl was dependent on UNG-1 and CHK-2. Methyl viologen, ndx-1 (RNAi) and ndx-2 (RNAi) enhanced the incidence of Pvl among exo-3 mutants only when combined with dut-1 (RNAi). This further increase in Pvl incidence was independent of NTH-1. These results indicate that EXO-3 prevents developmental delay and Pvl in C. elegans, which are induced via DNA glycosylase-initiated checkpoint activation.

The Base Excision Repair Pathway in the Nematode Caenorhabditis elegans

“…C. elegans strains mutated for apn-1 showed up to 5-fold increase in mutation rates as discerned by a gfp-lacZ reporter, which has an insertion that sets lacZ out of frame and when mutated brings lacZ in frame (Zakaria et al, 2010). Under normal growth conditions, these apn-1 mutants show no reduction in life span despite the increase burden of mutations (Zakaria et al, 2010;Miyaji et al, 2018). However, upon exposure to DNA damaging agents such as MMS and tert-butylhydroperoxide (t-BH), the apn-1 mutants displayed a reduction in life span by 3-4 days (Zakaria et al, 2010).…”

“…Despite deletion of the ung-1 gene, the resulting mutant strain demonstrated a normal egg-laying rate and larval development, in addition to a normal life span compared to N2 strain ( Nakamura et al, 2008 ). Interestingly, the C. elegans ung-1 mutant was no more sensitive than the wild type (WT) upon exposure to sodium hydrogen sulfite (NaHSO 3 ); a DNA damaging agent that induces cytosine base deamination and creates U ⋅ G lesion ( Burgers and Klein, 1986 ; Nakamura et al, 2008 ; Miyaji et al, 2018 ). These findings are unique to C. elegans as S. cerevisiae and E. coli cells lacking Ung activity showed high sensitivity to NaHSO 3 ( Simmons and Friedberg, 1979 ; Burgers and Klein, 1986 ).…”

“…Caenorhabditis elegans knockout and knockdown for the APN-1 and EXO-3 AP endonucleases do not lead to inviable phenotypes. The exo-3 mutant strains often demonstrate a more severe phenotype compared to apn-1 mutants ( Figure 2 ; Schlotterer et al, 2010 ; Zakaria et al, 2010 ; Kato et al, 2015 ; Miyaji et al, 2018 ). RNA interference (RNAi) studies indicate that exo-3 knockdown does not terminate growth completely, rather it results in developmental delay by an average of 6 h from the wild type strain; a delay that is aggravated by exposure to toxic agents such as MMS and NaHSO 3 ( Miyaji et al, 2018 ).…”

“…The exo-3 mutant strains often demonstrate a more severe phenotype compared to apn-1 mutants ( Figure 2 ; Schlotterer et al, 2010 ; Zakaria et al, 2010 ; Kato et al, 2015 ; Miyaji et al, 2018 ). RNA interference (RNAi) studies indicate that exo-3 knockdown does not terminate growth completely, rather it results in developmental delay by an average of 6 h from the wild type strain; a delay that is aggravated by exposure to toxic agents such as MMS and NaHSO 3 ( Miyaji et al, 2018 ). Deletion of apn-1 in addition to exo-3 did not alter the observed phenotype as exo-3;apn-1 double mutants were reported to demonstrate developmental delay as well ( Miyaji et al, 2018 ).…”

“…RNA interference (RNAi) studies indicate that exo-3 knockdown does not terminate growth completely, rather it results in developmental delay by an average of 6 h from the wild type strain; a delay that is aggravated by exposure to toxic agents such as MMS and NaHSO 3 ( Miyaji et al, 2018 ). Deletion of apn-1 in addition to exo-3 did not alter the observed phenotype as exo-3;apn-1 double mutants were reported to demonstrate developmental delay as well ( Miyaji et al, 2018 ). In addition, exo-3 deletion reduced C. elegans motility (head and overall body), and induces abnormal vulval organogenesis ( Schlotterer et al, 2010 ; Miyaji et al, 2018 ).…”