Molecular Biology

Molecular Biology SECTION F DNA damage, repair, and recombination F1 Mutagenesis (诱变） F2 DNA damage F3 DNA repair F4 Recombination link

Molecular Biology F1 Mutagenesis Mutation Replication fidelity Mutagens: chemical & physical Mutagenesis: direct & indirect

Molecular Biology Mutation （突变） Replication Fidelity (复制的忠实性） Mutagenesis Mutagens （诱变剂）

Molecular Biology Mutation-1 Concept:Permanent, heritablealterations in the base sequence of DNA Arise either through spontaneous errors in DNA replication or meiotic recombination or as a consequence of the damaging effects of physical or chemical agents on the DNA.

Molecular Biology Mutation-2 Point mutation: is the simplest mutation-a single base change. 1, Transition (转换): Purine or pyrimidine is replaced by the other AG T C 2, Transversion (颠换): a purine is replaced by a pyrimidine or vice verse A T or C T  A or G G T or C C  A or G

Phenotypic effects The sites Types • Noncoding DNA • Nonregulatory DNA • 3rd position of a codon No Silent mutation Missense mutation Yes or No Coding DNA  altered AA Coding DNA  stop codon  truncated protein Yes Nonsense mutation Molecular Biology Mutation-3 Effects of a point mutation

Molecular Biology Mutation-4 Insertions or deletions The addition or loss of one or more bases in a DNA region Frameshift mutations The ORF of a protein encoded gene is changed so that the C-terminal side of the mutation is completely changed.

Molecular Biology Examples of deletion mutations

Molecular Biology Replication fidelity The need to preserve the meaning of the genetic information from one generation to the next, so the error rate of DNA replication is much lower. E.g. Spontaneous errors in DNA replication is very rare, one error per 1010 base in E. coli. Molecular mechanisms of the replication fidelity • DNA polymerase: Watson-Crick base pairing • The occasional error can be detected and repaired by 3’ 5’ proofreading exonuclease. • RNA priming: proofreading the 5’ end of the lagging strand • Mismatch repair mechanism(F3)

Molecular Biology Proofreading by E. coli polymerase (Figure from Gene VII)

Molecular Biology Mutagens The physical and chemical agents of causing DNA damage that can be converted to mutations. 1, Physical mutagens High-energy ionizing radiation: x-rays and g-rays  strand breaks and base/sugar destruction (cause extensive chemical alterations) Nonionizing radiation: UV light pyrimidine dimers from adjacent 2, Chemical mutagens Base analogs: direct mutagenesis; Nitrous acid: deaminates C to produce U and guanine analog; Alkylating agents and Arylating agents produce lesions (indirect mutagenesis); Intercalators: insertion and deletion mutations

Molecular Biology Base analogs: derivatives of the normal bases with altered base pairing properties. Nitrous acid: deaminates C to produce U, resulting in G·C  A·U

Molecular Biology Mutagenesis Mutagenesis is the molecular process in which the mutation is generated, including direct and indirect mutagenesis.

OH AGCTTCCTA TCGAAGGAT Br Br H : G • Base analog • incorporation O enol form AGCTBCCTA TCGAAGGAT • 1st round • of replication AGCTTCCTA TCGAAGGAT AGCTBCCTA TCGAGGGAT H : A • 2nd round • of replication O Keto form AGCTBCCTA TCGAAGGAT AGCTCCCTA TCGAGGGAT 5-BrU Molecular Biology E.g. 1, Direct mutagenesis The stable, unrepaired base with altered base pairing properties in the DNA is fixed to a mutation during DNA replication. A·TG·C transition

Molecular Biology 2, Indirect mutagenesis The mutation is introduced as a result of an error-prone repair. Translesion DNA synthesis to maintain the DNA integrity but not the sequence accuracy: when damage occurs immediately ahead of an advancing fork, which is unsuitable for recombination repair (F4), the daughter strand is synthesized regardless of the the base identity of the damaged sites of the parental DNA. E。g。 E. coli translesion replication: SOS response

Molecular Biology F3 & F4 DNA damage and repair Physical and chemical Mutagens The lesions of extensive, right ahead of Replication Fork minor or moderate lesions DNA damage (lesions) Error-free Repairing Error-prone repairing mutations Completely repaired

Molecular Biology F2 DNA damage-1 DNA lesion is an alteration to the normal chemicalor physicalstructure of the DNA. 1,Oxidative damage (氧化损伤） DNA lesions (DNA损害） 2, Bulky adducts (加合物） • Occurs under Normal condition • Increased by • ionizing radiation • (physical mutagens) UV light (physical mutagens) Carcinogen (Chemical mutagens) 3，Alkylation (烷基化作用） Alkylating agents (Chemical mutagens)

Molecular Biology F2 DNA damage-2 The biological effect of the unrepaired DNA lesions The altered chemistry of the bases may lead to loss of base pairing or altered base pairing. If such a lesion was allowed to remain in the DNA, Physical distortion in the DNA Blocks replication And/or transcription Living cell A mutation could become fixed by direct or indirect mutagenesis Lethal (cell death) Mutagenic

Molecular Biology F2 DNA damage-3 Spontaneous DNA lesions Because of inherent chemical reactivity of the DNA and the presence of normal, reactive chemical species within the cell • Deamination (转氨作用）: CU; methylcytosine T, hard to be detected • Depurination (脱瞟呤作用) : break of the glycosylic bond, non-coding lesion. • Depyrimidine (脱嘧啶作用)

Oxidation products Molecular Biology F2 DNA damage-4 Oxidative damage 1, Is a kind of DNA lesions caused by reactive oxygen species such as superoxide and hydroxyl radicals, which occurs under normal conditions. 2, The level of this damage can be INCREEASED by hydroxyl radicals from the radiolysis of water caused by ionizing radiation.

Molecular Biology F2 DNA damage-5 Alkylation Alkylating agents are electrophilic chemicals which readily add alkyl (e.g. methyl) groups to various positions on nucleic acids distinct from those methylated by normal methylating enzymes such as methylmethane sulfonate （甲基甲烷磺酸盐）and ethylnitrosourea (乙基亚硝基脲).

Molecular Biology alkylating agents Alkylated bases

Molecular Biology F2 DNA damage-6 Bulky adducts A kind of DNA lesions that distort the double helix and cause localized denaturation, for example pyrimidine dimers and arylating agents adducts. These lesions disrupt the normal function of the DNA.

Molecular Biology Cyclobutane pyrimidine dimer(嘧啶二聚体) Guanine adduct of benzo[a]pyrene Aromatic arylating agents Covalent adducts

Molecular Biology F3 DNA repair-1 Photoreactivation Cyclobutane pyrimidine dimers can be monomerized again by DNA photolyases (photoreactivating enzymes) in the presence of visible light. Direct reversal of a lesion and is error-free

Molecular Biology F3 DNA repair-2 Alkyltransferase Removes the alkyl group from mutagenic O6-alkylguanine which can base-pair with T. The alkyl group is transferred to the protein itself and inactivate it. Direct reversal of a lesion and is error-free

Molecular Biology F3 DNA repair-3 Excision repair • There are two forms, nucleotide excision repair (NER) and base excision repair (BER). • Is a ubiquitous mechanism repairing a variety of lesions. • Error-free repair

Molecular Biology Nucleotide excision repair • An endonuclease cleaves DNA a precise number of bases on either sides of the lesions (in E.coliUvrABC endonulcease removes pyrimidine dimers) • Excised lesion-DNA fragment is removed • The gap is filled by DNA polymerase I and sealed by ligase

Molecular Biology Base excision repair • modified bases are recognized by relatively specific DNA glycosylases which cleave the N-glycosylic bond between the altered base and the sugar), leaving an apurinic or apyrimidinic (AP) site. • An AP endonuclease then cleaves the DNA at this site and a gap may be created by further exonuclease activity. The gap is generally larger in NER and can be as small as one nucleotide in BER. • The gap is filled by DNA polymerase I and sealed by ligase

Molecular Biology F3 DNA repair-4 Mismatch repair Is a specialized form of excision repair that deals with any base mispairs produced during replication and that have escaped proofreading. error-free

Molecular Biology The parental strand is methylated at N6 position of all As in GATC sites, but methylation of the daughter strand lag a few minutes after replication MutH/MutS recognize the mismatched base pair and the nearby GATC DNA helicase II, SSB, exonuclease I remove the DNA fragment including the mismatch DNA polymerase III & DNA ligase fill in the gap Expensive to keep the accuracy

Molecular Biology DNA Repair Mechanisms The proteins of DNA replication also play roles in the life-preserving repair mechanisms, helping to ensure the extract replication of template DNA.

Molecular Biology F4 Recombination Includes homologous recombination, site-specific recombination and transposition. Recombination is an important reason for variable DNA sequences among different populations of the same species.

Molecular Biology Homologous recombination ( 同源重组） Also known as general recombination, this process involves the exchange of homologous regions between two DNA molecules. 1, In diploid eukaryotes: commonly occurs during meiosis by crossing over. 2, In Haploidprokaryotes: recA-dependent, Holliday model 3,DNA repair in replication fork

Molecular Biology Haploid prokaryotes recombination Between the two homologous DNA duplex • between the replicated portions of a partially duplicated DNA • between the chromosomal DNA and acquired “foreign” DNA Holliday model

Molecular Biology recA-dependent bacterial homologous recombination • Homologous DNA pairs 2. Nicks made near Chi (GCTGGTGG) sites by a nuclease. 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3. ssDNA carrying the 5’ ends of the nicks is coated byRecA to form RecA-ssDNA dilaments.

Molecular Biology 4. RecA-ssDNA filaments search the opposite DNA duplex for corresponding sequence (invasion). 5. form a four-branched Holliday structure 6. Branch migration

Resolving Holliday junction Molecular Biology

Molecular Biology Recombination based DNA repair at replication fork, also called post-replication repair • a. Replication encounter a DNA lesion • b. Skip the lesion & re-initiate on the other side of the lesion • c. Fill the daughter strand gap by replacing it with the corresponding section from the parental sister strand by recombination • The original lesion can be removed later by normal excision repair.

Molecular Biology Site-specific recombination • Exchange of non-homologous but specific pieces of DNA • Mediated by proteins that recognize specific DNA sequences.

Molecular Biology bacteriophage λ insertion • Aλ-encoded integrase (Int): makes staggered cuts in the specific sites with 7 bp overhangs • Int and IHF (integration host factor encoded by bacteria): promote recombination between these sites and insertion of the λDNA into the host chromosome (prophage) • λ-encoded excisionase: excision of the phage DNA and the process reverse Site-specific recombinases such as FLP, Cre, and C31 have emerged as powerful tools to manipulate genomes of eukaryotic model organisms.

Molecular Biology Antibody diversity In eukaryotes, site-specific recombination is responsible for the generation of antibody diversity. Immunoglobulins are composed of two heavy (H) chains and two light (L) chains of various types, both of which contain regions of constant and variable amino acid sequence. The sequences of the variable regions of these chains in germ cells are encoded by three gene segments: V, D and J. There are a total of 250 V, 15 D and five J genes for H chains and 250 V and four J for L chains. Recombination between these segments during differentiation of antibody-producing cells can produce an enormous number (> 108) of different H and L gene sequences and hence antibody specificities.

Molecular Biology Transposition Transposons or transposable elements are small DNA sequences that can move to virtually any position in a cell’s genome. Transposition has also been called illegitimate recombination. 1, it requires no homology between sequences nor is it site-specific 2, it is relatively inefficient 3, Require Transposase encoded by the transposon ( 转座子）

Molecular Biology • Various transposons • In E. coli: • IS elements/insertion sequence, 1-2 kb in length, comprise a transposase gene flanked by a short inverted terminal repeats(ITR, ~20 bp) • Tn transposon series carry transposition elements and β-lactamase (penicillin resistance) • Eukaryotic transposons, many are retrotransposons: • Yeast Ty element encodes protein similar to RT (reverse transcriptase). • E.g. P-element, piggyBac transposon

Molecular Biology Simplified Transposition process

Molecular Biology