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

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.

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Base mismatches Copy Number Variations DNA LESION DNA REPAIR PATHWAY Nucleotide Excision Repair Base Excision Repair ICL Repair DSB Repair Mismatch Repair Figure 1: Genome maintenance pathways The genome is challenged constantly by a wide range of endogenous and exogenous DNA damaging agents. Many types of DNA lesions exist, such as abasic sites, bulky adducts, DNA strand breaks, base mismatches, and interstrand crosslinks. To avoid genome instability, five main DNA repair pathways exist: nucleotide excision repair, base excision repair, double strand break repair, mismatch repair, and DNA interstrand crosslink repair. 22 Chapter 1 Interstrand Crosslink RepairDNA-interstrand crosslinks (ICLs) are DNA lesions that covalently bind the two strands of the double helix 76 . Because ICLs block strand separation, which is required for DNA replication and transcription, they are among the most toxic DNA lesions. ICL-inducing agents are effective in chemotherapy because they preferentially affect rapidly-dividing cells, such as cancer cells 77 . ICL inducing agents Exogenous sources of ICLsMany drugs used for chemotherapy induce ICLs, such as cisplatin and its derivatives, mitomycin C (MMC), deoxybutane (DEB), psoralens, and nitrogen mustards. These agents cause a multitude of DNA adducts, and ICLs represent just a small percentage of the lesions formed 78 . Yet, ICLs are considered the main cause of toxicity of these drugs. Although all ICLs are formed by crosslinking the two strands of the DNA, the chemical and structural properties of the various ICLs differ widely. First, the sequence context in which an ICL can form depends on the chemistry of the ICLinducing agent. For example, cisplatin and MMC induce ICLs in a 5' CG context, while psoralens act in a 5' TA sequence 78 . Secondly, the structure of the ICL and the resulting distortion of the DNA double helix differs depending on the ICL-inducing agent (Figure 5). Cisplatin causes a major distortion of the double helix, forcing the bases opposite the crosslinked Gs to be extrahelical. On the other hand, MMC, psoralens, and nitrogen mustards cause minor distortions in the DNA structure 78 . Endogenous sources of ICLsStudying ICLs formed endogenously in cells is challenging due to the scarcity of these lesions and the lack of detection methods. However, both in vivo and in vitro studies identified aldehydes as a possible endogenous source of ICLs. Aldehydes are a highly reactive class of endogenous metabolites that can form a variety of DNA adducts, including ICLs 79 (Figure 5). Acetaldehyde is a product of ethanol oxidation and the metabolism of carbohydrates, and can form ICLs between guanines 80,81 . Formaldehyde is an aldehyde that is highly concentrated in human plasma and is formed during various cellular processes such as dealkylation of methylated DNA and histone demethylation [81][82][83] . Other ICL-inducing aldehydes are generated through lipid peroxidation, such as acrolein, malondialdehyde, crotonaldehyde, and 4-hydroxynonenal 84 .In chapter 2 we show that ...