1Transcription-coupled repair (TCR) removes base damage on the transcribed strand of a gene to 2 ensure a quick resumption of transcription. Based on the absence of key enzymes for TCR and 3 empirical evidence, TCR was thought to be missing in Drosophila melanogaster. The recent 4 demonstration of TCR in S2 cells raises the question about the involved genes. Since the 5 mismatch repair (MMR) pathway serves a central role in TCR, at least in Escherichia coli, we 6 studied the mutational signatures in flies with a deletion of the MMR gene spellchecker1 (spel1), 7 a MutS homolog. Whole-genome sequencing of mutation accumulation (MA) lines obtained 7,345 8 new single nucleotide variants (SNVs) and 5,672 short indel mutations, the largest data set from 9 an MA study in D. melanogaster. Based on the observed mutational strand-asymmetries, we 10 conclude that TCR is still active without spel1. The operation of TCR is further confirmed by a 11 negative association between mutation rate and gene expression. Surprisingly, the TCR 12 signatures are detected for introns, but not for exons. We propose that an additional exon-specific 13 repair pathway is masking the signature of TCR. This study presents the first step towards 14 understanding the molecular basis of TCR in Drosophila melanogaster. 15

1Sex biases in mutation rates may affect the rate of adaptive evolution. In many species, males 2 have higher mutation rates than females when single nucleotide variants (SNVs) are considered. 3In contrast, indel mutations in humans and chimpanzees are female-biased. In Drosophila 4 melanogaster, direct estimates of mutation rates did not uncover sex differences, but a recent 5 analysis suggested the presence of male-biased SNVs mutations. Here we study the sex-specific 6 mutation processes using mutation accumulation data from mismatch-repair deficient D. 7 melanogaster. We find that sex differences in flies are similar to the ones observed in humans: a 8 higher mutation rate for SNVs in males and a higher indel rate in females. These results have 9 major implications for the study of neutral variation and adaptation in Drosophila. 10 192