Rev1-dependent TLS and ggNER converge on helix-distorting oxidative DNA lesions resulting from progressive mitochondrial dysfunction (see alsosupplemental Figure 6). We investigated the involvement of Rev1-dependent TLS at helix-distorting lipid peroxidation–derived nucleotide adduct by treating Rev1 HSCs with a radical scavenger, by using an in cellulo TLS assay, by investigating the sensitivity of Rev1 cells to a lipid peroxidation–derived aldehyde, to oxidative stress, and by measuring oxidative stress in bone marrow. We then characterized the quantity and functionality of Rev1Xpc mitochondria. *P < .05; **P < .01; ***P < .001; ****P < .0001. Data are mean ± SEM. (A) DNA breaks (γH2AX) in cultured HSCs (LSK-SLAM), WT (n = 3), and Rev1 (n = 3), treated or nontreated with the ROS scavenger NAC. The fraction of positive cells shown was normalized relative to WT. (B) The prototypic DNA-reactive lipid peroxidation–derived aldehyde 4-ONE and its adduction to a cytosine base (H-εdC). (C) Top: TLS assay at a site-specific H-εdC. MEFs were transfected with the substrate, followed by incubation to allow TLS, and by recovery of covalently closed progeny plasmids in Escherichia coli. The fraction of recovered substrate, compared with an undamaged internal control, is a measure of TLS activity of the MEFs. Bottom: Relative efficiency and mutation spectrum of TLS events at a site-specific H-εdC lesion. (D) Clonal survival of WT, Rev1, Xpc, and Rev1Xpc MEFs in response to the addition of the mitochondrial poison paraquat to the growth medium. (E) Clonal survival of Xpc and Rev1Xpc MEFs in response to the addition of 4-HNE to the growth medium. Oxidative stress in the bone marrow of Rev1Xpc mice as evidenced by: lipofuscin accumulation (brown inclusions) in bone marrow of moribund mice (F), 4-HNE-positive cells (G), activation of p38 signaling [phospho (γ)p38 staining] (H), and accumulation of free radical-induced oxidative DNA lesions (OHdG-positive cells) (I) in Rev1Xpc mice: 1m (n = 3-4), 3m (n = 3-5), MB (n = 5-6). The fraction of positive cells shown was normalized relative to 3-month-old WT bone marrow. (J) Relative mtDNA contents, as determined by real-time PCR, in bone marrow from Xpc (n = 5-6) and Rev1Xpc (n = 5-6) mice. All mtDNA levels were normalized to those in 3-month-old Xpc mice. (K) Western blot of mitochondrial complexes I to IV in bone marrow from Xpc and Rev1Xpc mice (4 mice per group). Lamin B1: internal standard. (L) Expression of the mitochondrial stress proteins UCP2 and PGC-1α in WT, Xpc, Rev1, and Rev1Xpc bone marrow. Lamin B: internal standard. wk, weeks; m, months; MB, moribund (see Figure 2C for survival data). (M) Mitochondrial membrane potentials in bone marrow of Xpc and Rev1Xpc mice. All potentials in Rev1Xpc bone marrow were normalized to those in Xpc bone marrow of the same age. 1 m (n = 4), 1.5 m (n = 4), 3 m (n = 4-6), MB (n = 3-4). (N) Basal oxygen consumption rates in viable cells from bone marrow from Xpc and Rev1Xpc mice. All oxygen consumption rates in Rev1Xpc bone marrow were normalized to those in Xpc bone marrow of the same age. 2wk (n = 5-6), 3 m (n = 3).