SECTION F DNA damage, repair, and recombination F1 Mutagenesis ( 诱变） F2DNA damage F3 DNA repair F4Recombination

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

Mutation Replication Fidelity ( 复制的忠实 性） Mutagens Mutagenesis （突变） （诱变剂）

Mutation-1 Concept: Permanent, heritable alterations 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.

1-Transition ( 转换 ): Purine or pyrimidine is replaced by the other A  GT  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 Point mutation: is the simplest mutation-a single base change. Mutation-2

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

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

Examples of deletion mutations

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 base in E. coli.

Molecular mechanisms of the replication fidelity 1.DNA polymerase: Watson-Crick base pairing 2.The occasional error can be detected and repaired by 3’  5’ proofreading exonuclease. 3.RNA priming: proofreading the 5’ end of the lagging strand 4.Mismatch repair mechanism(F3)

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

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  -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

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

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

1, Direct mutagenesis The stable, unrepaired base with altered base pairing properties in the DNA is fixed to a mutation during DNA replication. 5-BrU : G : A enol form Br OH H O Br Keto form H O AGCTTCCTA TCGAAGGAT AGCTBCCTA TCGAAGGAT 1.Base analog incorporation AGCTBCCTA TCGAGGGAT AGCTTCCTA TCGAAGGAT 1st round of replication AGCTBCCTA TCGAAGGAT AGCTCCCTA TCGAGGGAT 3.2nd round of replication E.g. A·T  G·C transition

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

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

F2DNA damage-1 DNA lesions (DNA 损害） 1, Oxidative damage ( 氧化损伤） 3 ， Alkylation ( 烷基化作用） 2, Bulky adducts ( 加合物） 1.Occurs under Normal conditionOccurs under Normal condition 2.Increased byIncreased by ionizing radiation (physical mutagens) Alkylating agents (Chemical mutagens) UV light (physical mutagens) Carcinogen (Chemical mutagens) DNA lesion is an alteration to the normal chemical or physical structure of the DNA.

The biological effect of the unrepaired DNA lesions Lethal (cell death) Physical distortion in the DNA Blocks replication And/or transcription Mutagenic A mutation could become fixed by direct or indirect mutagenesis Living cell 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, F2DNA damage-2

Spontaneous DNA lesions Because of inherent chemical reactivity of the DNA and the presence of normal, reactive chemical species within the cell 1.Deamination ( 转氨作用） : C  U; methylcytosine  T, hard to be detected 2.Depurination ( 脱瞟呤作用 ) : break of the glycosylic bond, non-coding lesion. 3.Depyrimidine ( 脱嘧啶作用 ) F2DNA damage-3

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. Oxidative damage F2 DNA damage-4 Oxidation products

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 ). Alkylation F2DNA damage-5

alkylating agents Alkylated bases

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. Bulky adducts F2DNA damage-6

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

Photoreactivation F3DNA repair-1 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

Removes the alkyl group from mutagenic O 6 - alkylguanine which can base-pair with T. The alkyl group is transferred to the protein itself and inactivate it. Alkyltransferase F3DNA repair-2

1.There are two forms, nucleotide excision repair (NER) and base excision repair (BER). 2.Is a ubiquitous mechanism repairing a variety of lesions. 3.Error-free repair Excision repair F3DNA repair-3

Nucleotide excision repair 1.An endonuclease cleaves DNA a precise number of bases on either sides of the lesions (in E.coli UvrABC endonulcease removes pyrimidine dimers) 2.Excised lesion-DNA fragment is removed 3.The gap is filled by DNA polymerase I and sealed by ligase

Base excision repair 1.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. 2.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. 3.The gap is filled by DNA polymerase I and sealed by ligase

Is a specialized form of excision repair that deals with any base mispairs produced during replication and that have escaped proofreading. error-free Mismatch repair F3DNA repair-4

The parental strand is methylated at N 6 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

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.

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

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

Haploid prokaryotes recombination Between the two homologous DNA duplex 1. between the replicated portions of a partially duplicated DNA 2.between the chromosomal DNA and acquired “foreign” DNA Holliday model

2.Nicks made near Chi (GCTGGTGG) sites by a nuclease. 3. ssDNA carrying the 5’ ends of the nicks is coated by RecA to form RecA-ssDNA dilaments. recA-dependent bacterial homologous recombination 1.Homologous DNA pairs 3’5’ 3’5’ 3’ 5’

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

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 d.The original lesion can be removed later by normal excision repair.

Site-specific recombination 1.Exchange of non-homologous but specific pieces of DNA 2.Mediated by proteins that recognize specific DNA sequences.

1.A λ-encoded integrase (Int): makes staggered cuts in the specific sites with 7 bp overhangs 2.Int and IHF (integration host factor encoded by bacteria): promote recombination between these sites and insertion of the λ DNA into the host chromosome (prophage) 3.λ-encoded excisionase: excision of the phage DNA and the process reverse bacteriophage λ insertion Site-specific recombinases such as FLP, Cre, and C31 have emerged as powerful tools to manipulate genomes of eukaryotic model organisms.

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.

Antibody diversity 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 (> 10 8 ) of different H and L gene sequences and hence antibody specificities.

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）

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

Simplified Transposition process