Molecular Biology Course SECTION F DNA damage, repair, and recombination link

F1F1 Mutagenesis ( 诱变） F1F1 Mutagenesis ( 诱变） DNA damage, repair & recombination F2F2DNA damage F2F2DNA damage Mutation: replication fidelity, mutagens, mutagenesis F3F3 DNA repair F3F3 DNA repair DNA lesions: oxidative damage, alkylation, bulky adducts Photoreaction, alkyltransferase, excision repair, mismatch repair, hereditary repair defects Homologous recombination, site-specific recombination, transposition F4F4Recombination F4F4Recombination

DNA damage, repair & recombination F1F1 Mutagenesis ( 诱变） F1F1 Mutagenesis ( 诱变） Mutation Replication fidelity Mutagens: chemical & physical Mutagenesis: direct & indirect

Mutation Replication Fidelity ( 复制的精确 性） Mutagens Mutagenesis （突变） （诱变剂） back

F1-1 Mutation F1 Mutaagenesis Permanent, heritable alterations in the base sequence of DNA Reasons 1.Spontaneous errors in DNA replication or meiotic recombination 2.A consequence of the damaging effects of physical or chemical mutagens on DNA

TransitionTransition : Purine or pyrimidine is replaced by the other A  GT  C Transversion: a purine is replaced by a pyrimidine or vice verseTransversion A  T or C T  A or G G  T or C C  A or G Point mutation (a single base change) F1 Mutaagenesis

Noncoding DNA Nonregulatory DNA 3 rd position of a codon Silent mutation Coding DNA  altered AA Missense mutation Phenotypic effects No Coding DNA  stop codon  truncated protein Nonsense mutation Effects of a point mutation F1 Mutaagenesis Yes or No Yes

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 F1 Mutaagenesis

Examples of deletion mutations

F1-2 Replication fidelity F1 Mutaagenesis Mutation relevant 1.Spontaneous errors in DNA replication is very rare, one error per base in E. coli. Important for preserve the genetic information from one generation to the next

Molecular mechanisms for the replication fidelity 1.DNA polymerase: Watson-Crick base pairing 2.3’  5’ proofreading exonuclease. 3.RNA priming: proofreading the 5’ end of the lagging strand 4.Mismatch repair (F3) F1 Mutaagenesis

by E. coli polymerase Proofreading F1 Mutaagenesis

F1-3&4: Mutagens F1 Mutaagenesis Mutation relevant Cause DNA damage that can be converted to mutations.

Physical mutagens High-energy ionizing radiation: X-rays and g-rays  strand breaks and base/sugar destruction Nonionizing radiation : UV light  pyrimidine dimers Chemical mutagens Base analogs: direct mutagenesis Nitrous acid: deaminates C to produce U Alkylating agents Arylating agents Lesions-indirect mutagenesis (F2) F1 Mutaagenesis

Base analogs: derivatives of the normal bases incorporated in DNA, altering base pairing properties. Nitrous acid: deaminates C to produce U, resulting in G·C  A·U

F1-3&4: Mutagenesis F1 Mutaagenesis The molecular process in which the mutation is generated. Note: the great majority of lesions introduced by chemical and physical mutagens are repaired by one or more of the error-free DNA repair mechanisms before the lesions is encounter by a replication fork

Direct mutagenesis The stable, unrepaired base with altered base pairing properties in the DNA is fixed to a mutation during DNA replication. F1 Mutaagenesis

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 2.1st round of replication AGCTBCCTA TCGAAGGAT AGCTCCCTA TCGAGGGAT 3.2nd round of replication A·T  G·C transition F1 Mutaagenesis

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. F1 Mutaagenesis

E. coli translession replication: SOS response: Higher levels of DNA damage effectively inhibit DNA replication and trigger a stress response in the cell, involving a regulated increase (induction) in the levels of a number of proteins. This is called the SOS response. 1.Some of the induced proteins, such as the UvrA and UvrB proteins, have roles in normal DNA repair pathways. 2.A number of the induced proteins, however, are part of a specialized replication system that can REPLICATE PAST the DNA lesions that block DNA polymerase III. back F1 Mutaagenesis

Proper base pairing is often impossible and not strictly required at the site of a lesion because of the SOS response proteins, this translesion replication is error-prone. The resulting increase in mutagenesis does not contradict the general principle that replication accuracy is important (the resulting mutations actually kill many cells). This is the biological price that is paid, however, to overcome the general barrier to replication and permit at least a few mutant cells to survive.

DNA damage and repair Mutagen ( 诱变剂） Completely repaired DNA damage (lesions) chemical reactivity of the bases Error-free Repairing mutations Indirect mutagenesis F DNA damage, repair and recombination minor or moderate Extensive, right before R eplication F ork (not repairable) Direct mutagenesis

DNA damage, repair & recombination F2F2DNA damage F2F2DNA damage DNA lesions: oxidative damage Alkylation bulky adducts

DNA lesions (DNA 损害） Oxidative damage ( 氧化损伤） Alkylation ( 烷基化作用） 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 damage, repair & recombination

F2-1DNA lessions 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 of the local DNA structure Blocks replication And/or transcription Mutagenic Allowed to Remained in the DNA A mutation could become fixed by direct or indirect mutagenesis Living cell Altered chemistry of the bases DNA lessions

Spontaneous DNA lesions 1.Inherent chemical reactivity of the DNA 2.The presence of normal, reactive chemical species within the cell 1.Deamination ( 转氨作用） : C  UC  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 ) back

Chemical reactivity of bases is responsible for some DNA lesion DNA lessions

deamination --ATGCTACG-- --TACGATGC-- --ATGUTACG-- --TACGATGC-- --ATG TACG-- --TACGATGC-- U --ATGCTACG-- --TACGATGC-- Uracil DNA glycosylase Cytosine deamination and repair back

DNA damage, repair & recombination F2-2Oxidative damage DNA lesions caused by reactive oxygen species such as superoxide and hydroxyl radicals

Oxidation products 1. occurs under NORMAL conditions in all aerobic cells due to the presence of reactive oxygen species (ROS), such as superoxide, hydrogen peroxide, and the hydroxyl radicals (OH). 2.The level of this damage can be INCREEASED by hydroxyl radicals from the radiolysis of H 2 O caused by ionizing radiation DNA damage

DNA damage, repair & recombination F2-3Alkylation Nucleotide modification caused by electrophilic alkylating agents such as methylmethane sulfonate （甲基甲烷磺酸盐） and ethylnitrosourea ( 乙基亚硝基脲 )

Alkylated bases 1.Electrophilic chemicals adds alkyl groups to various positions on nucleic acids 2.Distinct from those methylated by normal methylating enzymes. alkylating agents

DNA damage, repair & recombination F2-4Bulky adducts 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

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

DNA damage, repair & recombination F3F3DNA repair F3F3DNA repair Photoreactivation ( 光活化作用 ) Alkyltransferase ( 烷基转移酶 ) Exision repair ( 切割修复 ) Mismatch repair ( 错配修复 ) Hereditary repair defects ( 遗传修复缺陷 )

DNA damage, repair & recombination F3-1: Photoreactivation Monomerization of cyclobutane pyrimidine dimers by DNA photolyases in the presence of visible light Direct reversal of a lesion and is error-free

DNA damage, repair & recombination F3-2: Alkyltransferase 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.

The response is adaptive because it is induced in E. coli by low levels of alkylating agents and gives increased protection against the lethal and mutagenic effects of the high doses DNA repair

DNA damage, repair & recombination F3-3: Excision repair 1.Includs nucleotide excision repair (NER) and base excision repair (BER). 2.Is a ubiquitous mechanism repairing a variety of lesions. 3.Error-free repair

Nucleotide excision repair DNA repair 1.An endonuclease cleaves DNA a precise number of bases on both sides of the lesions (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 DNA glycolases cleaves apurinic or pyrimidine site DNA polymerase DNA ligase DNA repair cleaves N-glycosylic bond AP endonuclease 3’  5’ cleavage and & 5’  3’ synthesis

DNA damage, repair & recombination F3-3: Mismatch repair A specialized form of excision repair which deals with any base mispairs produced during replication and which have escaped proofreading error-free

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 back

DNA damage, repair & recombination Homologous recombination Site-specific recombination Transposition F4F4Recombination F4F4Recombination Mutation Relevance An important reason for variable DNA sequences among different populations of the same species

F4-1 Homologous recombination ( 同源重组） Diploid eukaryotes: crossing over Haploid prokaryotes: recA-dependent, Holliday model DNA repair in replication fork DNA damage, repair & recombination The exchange of homologous regions between two DNA moleculs

Diploid eukaryotesDiploid eukaryotes: crossing over 1.Homologous chromosomes line up in meiosis (when) 2.The nonsister chromatids exchange equivalent sections (what) F4 Recombination

Haploid prokaryotesHaploid prokaryotes recombination Between the two homologous DNA duplex (where) 1. between the replicated portions of a partially duplicated DNA 2.between the chromosomal DNA and acquired “foreign” DNA Holliday model (How) F4 Recombination

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 F4 Recombination 1.Homologous DNA pairs 3’5’ 3’5’ 3’ 5’

3.RecA-ssDNA filaments search the opposite DNA duplex for corresponding sequence (invasion). 4.form a four-branched Holliday structurefour-branched Holliday structure 5.Branch migration back F4 Recombination

6.Resolving Holliday junctionesolving Holliday junction F4 Recombination

RuvAB is an asymmetric complex that promotes branch migration of a Holliday junction. F4 Recombination

Recombination based DNA repair at replication fork a.Replication encounter a DNA lesion b.Skip the lesion & re- initiate on the side of the lesion c.Fill the daughter strand gap by replacing it with the corresponding section from the parental sister strand d.post-replication repair of the left lesion

F4-2: Site-specific recombination ( 位点专一重组） DNA damage, repair & recombination 1.Exchange of non-homologous but specific pieces of DNA (what) 2.Mediated by proteins that recognize specific DNA sequences. (how)

1.l-encoded integrase (Int): makes staggered cuts in the specific sites 2.Int and IHF (integration host factor encoded by bacteria): recombination and insertion 3.l-encoded excisionase (XIS): excision of the phage DNA Site-specific recombination: bacteriophage  insertion Site-specific recombination: bacteriophage  insertion F4 Recombination

Site-specific recombination: Antibody diversity Site-specific recombination: Antibody diversity H and L are all encoded by three gene segments: V, D, J VDJ Two heavy chains (H) Two light chains (L) 2504 Enormous number (>10 8 ) of different H and L gene sequences can be produced by such a recombination F4 Recombination

F4-3 Transposition ( 转座作用） back DNA damage, repair & recombination 1.Requires no homology between sequences nor site-specific 2.Relatively inefficient 3.Require Transposase encoded by the transposon ( 转座 子）

Various transposons: In E. coli: IS elements/insertion sequence, 1-2 kb, comprise a transposase gene flanked by a short inverted terminal repeats Tn transposon series carry transposition elements and b-lactamase (penicillin resistance) Eukaryotic transposons, many are retrotransposons: Yeast Ty element encodes protein similar to RT (reverse transcriptase) F4 Recombination

Simplified Transposition process F4 Recombination