Acetaldehyde is a highly reactive, DNA damaging metabolite, produced upon alcohol consumption 1. Impaired acetaldehyde detoxification is common in the Asian population, and is associated with alcohol related cancers 1,2. Cellular protection against acetaldehyde-induced damage is provided by DNA crosslink repair; when impaired this causes Fanconi anaemia (FA), a disease resulting in failed blood production and cancer predisposition 3,4. Strikingly, combined inactivation of acetaldehyde detoxification and the FA pathway induces mutation, accelerates malignancies and causes the rapid attrition of blood stem cells 5-7. A key question concerns the nature of DNA damage caused by acetaldehyde, and how this is repaired. Here we generate acetaldehyde-induced DNA interstrand crosslinks (AA-ICLs) and determine their repair mechanism in Xenopus egg extract. We discover that two replication-coupled pathways repair these lesions. The first is the FA pathway, that operates using excision, analogous to the mechanism used for chemotherapeutic crosslinks caused by cisplatin. Yet, this AA-ICL repair results in elevated mutation frequency and altered mutational spectrum. The second repair modality requires replication fork convergence but unexpectedly does not involve DNA incisions, instead the acetaldehyde-crosslink itself is broken. The Y-family DNA polymerase REV1 completes repair, culminating in a distinct mutation spectrum. This work defines how DNA interstrand crosslinks caused by an endogenous and alcohol-derived metabolite are repaired, identifying an excision-independent mechanism. To study the repair of alcohol-induced DNA damage, we generated an acetaldehyde-crosslinked DNA substrate. Acetaldehyde reacts with guanine creating a crosslink precursor, N2-propanoguanine (PdG) (Fig. 1a) 8. In a 5'-CpG sequence, PdG reacts with the N2-amine of guanine on the opposite strand to create an interstrand acetaldehyde crosslink (AA-ICL). The crosslink exists in equilibrium between three states 9. We synthesized a site-specific native AANAT-ICL within an oligonucleotide duplex (Extended Data Fig. 1a, b, d, Supplementary Information Fig. 1). A control reaction of PdG with deoxyinosine (dIno), lacking an N2-amine, did not crosslink, confirming AANAT-ICL site-specificity (Extended Data Fig. 1c, for gel source data see Supplementary Information Fig. 2). AANAT-ICLs were stable at physiological pH and temperature (< 10% reversal after 72 h at 37 C) (Extended Data Fig. 1e). Elevated temperature (55 C) or acid did however reverse AANAT-ICL, consistent with Schiff base Top strand Unhooked Bottom strand Unhooked

Insertions and deletions (InDels) are frequently observed in natural protein evolution, yet their potential remains untapped in laboratory evolution. Here we introduce a transposonbased mutagenesis approach (TRIAD) to generate libraries of random variants with short inframe InDels, and screen TRIAD libraries to evolve a promiscuous arylesterase activity in a phosphotriesterase. The evolution exhibits features that differ from previous point mutagenesis campaigns: while the average activity of TRIAD variants is more compromised, a larger proportion has successfully adapted for the activity. Different functional profiles emerge: (i) both strong and weak trade-off between activities are observed; (ii) trade-off is more severe (20-to 35-fold increased k cat /K M in arylesterase with 60-400-fold decreases in phosphotriesterase activity) and (iii) improvements are present in k cat rather than just in K M , suggesting adaptive solutions. These distinct features make TRIAD an alternative to widely used point mutagenesis, accessing functional innovations and traversing unexplored fitness landscape regions.

The combination of ultrahigh-throughput screening and sequencing informs on function and intragenic epistasis within combinatorial protein mutant libraries. Establishing a droplet-based, in vitro compartmentalised approach for robust expression and screening of protein kinase cascades (>107 variants/day) allowed us to dissect the intrinsic molecular features of the MKK-ERK signalling pathway, without interference from endogenous cellular components. In a six-residue combinatorial library of the MKK1 docking domain, we identified 29,563 sequence permutations that allow MKK1 to efficiently phosphorylate and activate its downstream target kinase ERK2. A flexibly placed hydrophobic sequence motif emerges which is defined by higher order epistatic interactions between six residues, suggesting synergy that enables high connectivity in the sequence landscape. Through positive epistasis, MKK1 maintains function during mutagenesis, establishing the importance of co-dependent residues in mammalian protein kinase-substrate interactions, and creating a scenario for the evolution of diverse human signalling networks.