Structural and biochemical analysis of the UvrA-binding module of the bacterial transcription-repair coupling factor Mfd

Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet. Structural basis for transcription coupled repair: the N-terminus of Mfd resembles UvrB with degenerate ATPase motifs. Journal of Molecular Biology 355(4): 675-683. "[One] topic we touched on was mutation ... We totally missed the possible role of … [DNA] repair although … I later came to realise that DNA is so precious that probably many distinct repair mechanisms would exist. Nowadays, one could hardly discuss mutation without considering repair. " Francis Crick in "The double helix: a personal view" (Crick, 1974). 1.1 DNA repair DNA, the carrier of genetic information, is constantly threatened by a variety of damaging agents. Sources of DNA damage can be either exogenous (like chemicals or radiation) or endogenous (reactive metabolites like oxygen radicals or replication errors). They affect either the nucleobases or the backbone of the DNA helix (Lodish et al., 2000; Hoeijmakers, 2001). Examples for common DNA lesions are listed in table 1.1. DNA damage types Examples Caused by Base modifications Oxidation: 8-oxoguanine Alkylation: 7-methylguanine Deamination of cytosine to uracil Oxygen radicals Alkylating reagents Spontaneous deamination Mismatches G/T or A/C pairs Replication errors Breaks in the backbone Single strand breaks (SSBs) Double strand breaks (DSBs) Ionizing radiation or chemicals Bulky photoadducts Cyclobutane-pyrimidine dimers (CPDs), 6-4-Photoproducts UV radiation Cross-links Intrastrand cross-links Interstrand cross-links Cross-linking agents (bifunctional alkyklating agents) If left unrepaired, these DNA lesions can lead to mutations – which may in higher organisms result in cancer – or cell death. DNA damaging agents are often used as chemotherapeutics in cancer therapy in order to inhibit DNA replication and therefore stop cell division. In particular, DNA cross-linking agents, e.g cis-diammine dichloroplatinum(II) (cisplatin) or mitomycin C, are applied Cells have evolved multiple repair mechanisms, which use different enzymes and deal with different kinds of lesions (see table 1. humans, several inherited disorders were found to be associated with defects in DNA Introduction 2 damage repair genes (see chapters 1.1.1.2. and 1.1.2.5.). Many of these syndromes are characterized by premature ageing and cancer predispositions (Hoeijmakers, 2001). Repair mechanism Repair systems Applied to Direct damage reversal Photoreactivation Oxidative demethylation Ligation of SSBs Photoproducts (CPDs) Alkylated bases SSBs Damage removal (Excision repair) Nucleotide excision repair (NER)-Global genome repair (GGR)-Transcription coupled repair (TCR) Base excision repair (BER) Mismatch repair (MMR) Bulky, helix-distorting lesions like photoproducts, cisplatin-adducts, or cross-links Modified bases Single-base mispairs Double strand break (DSB) repair Homologous recombination (HR) Non-homologous end-joining …