3 DNA Replication Mutation Repair a DNA replication

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3. DNA Replication, Mutation, Repair a). DNA replication i). Cell cycle/ semi-conservative replication ii).

3. DNA Replication, Mutation, Repair a). DNA replication i). Cell cycle/ semi-conservative replication ii). Initiation of DNA replication iii). Discontinuous DNA synthesis iv). Components of the replication apparatus b). Mutation i). Types and rates of mutation ii). Spontaneous mutations in DNA replication iii). Lesions caused by mutagens c). DNA repair i). Types of lesions that require repair ii). Mechanisms of repair Proofreading by DNA polymerase Mismatched repair Excision repair iii). Defects in DNA repair or replication

The mammalian cell cycle DNA synthesis and histone synthesis Rapid growth and preparation for

The mammalian cell cycle DNA synthesis and histone synthesis Rapid growth and preparation for DNA synthesis S phase G 0 Quiescent cells G 1 phase G 2 M phase Mitosis phase Growth and preparation for cell division

DNA replication is semi-conservative Parental DNA strands Each of the parental strands serves as

DNA replication is semi-conservative Parental DNA strands Each of the parental strands serves as a template for a daughter strand Daughter DNA strands

Origins of DNA replication on mammalian chromosomes origins of DNA replication (every ~150 kb)

Origins of DNA replication on mammalian chromosomes origins of DNA replication (every ~150 kb) 5’ 3’ 3’ 5’ bidirectional replication bubble fusion of bubbles 5’ 3’ daughter chromosomes 3’ 5’

Initiation of DNA synthesis at the E. coli origin (ori) origin DNA sequence 5’

Initiation of DNA synthesis at the E. coli origin (ori) origin DNA sequence 5’ 3’ A A 3’ 5’ A binding of dna. A proteins A A DNA melting induced by the dna. A proteins A A dna. A proteins coalesce dna. B and dna. C proteins bind to the single-stranded DNA A A A B C dna. B further unwinds the helix

dna. G (primase) binds. . . A A A G A B C A

dna. G (primase) binds. . . A A A G A B C A A dna. B further unwinds the helix and displaces dna. A proteins A A A B C . . . and synthesizes an RNA primer G RNA primer

G B C 5’ Primasome dna B (helicase) dna C dna G (primase) template

G B C 5’ Primasome dna B (helicase) dna C dna G (primase) template strand 3’ OH 5’ RNA primer (~5 nucleotides) 3’

DNA polymerase 5’ 5’ RNA primer 5’ newly synthesized DNA 3’ 3’

DNA polymerase 5’ 5’ RNA primer 5’ newly synthesized DNA 3’ 3’

DNA Reaction catalyzed by DNA polymerase • all DNA polymerases require a primer with

DNA Reaction catalyzed by DNA polymerase • all DNA polymerases require a primer with a free 3’ OH group • all DNA polymerases catalyze chain growth in a 5’ to 3’ direction • some DNA polymerases have a 3’ to 5’ proofreading activity

Discontinuous synthesis of DNA 5’ 3’ 3’ 5’ 5’ 3’ Because DNA is always

Discontinuous synthesis of DNA 5’ 3’ 3’ 5’ 5’ 3’ Because DNA is always synthesized in a 5’ to 3’ direction, synthesis of one of the strands. . . 5’ 3’. . . has to be discontinuous. This is the lagging strand. 3’ 5’

Each replication fork has a leading and a lagging strand leading strand (synthesized continuously)

Each replication fork has a leading and a lagging strand leading strand (synthesized continuously) replication fork 5’ 3’ 3’ 5’ lagging strand (synthesized discontinuously) • The leading and lagging strand arrows show the direction of DNA chain elongation in a 5’ to 3’ direction • The small DNA pieces on the lagging strand are called Okazaki fragments (100 -1000 bases in length)

RNA primer direction of leading strand synthesis 3’ 5’ replication fork 5’ 3’ 3’

RNA primer direction of leading strand synthesis 3’ 5’ replication fork 5’ 3’ 3’ 5’ direction of lagging strand synthesis

Movement of the replication fork 5’ 3’

Movement of the replication fork 5’ 3’

Movement of the replication fork 5’ RNA primer Okazaki fragment RNA primer

Movement of the replication fork 5’ RNA primer Okazaki fragment RNA primer

3’ RNA primer 5’ pol III 5’ DNA polymerase III initiates at the primer

3’ RNA primer 5’ pol III 5’ DNA polymerase III initiates at the primer and elongates DNA up to the next RNA primer 3’ 3’ 5’ newly synthesized DNA (100 -1000 bases) (Okazaki fragment) 5’ 5’ pol I DNA polymerase I inititates at the end of the Okazaki fragment and further elongates the DNA chain while simultaneously removing the RNA primer with its 5’ to 3’ exonuclease activity

3’ 5’ newly synthesized DNA (Okazaki fragment) DNA ligase seals the gap by catalyzing

3’ 5’ newly synthesized DNA (Okazaki fragment) DNA ligase seals the gap by catalyzing the formation of a 3’, 5’-phosphodiester bond in an ATP-dependent reaction 3’ 5’

Proteins at the replication fork in E. coli Rep protein (helicase) 3’ 5’ pol

Proteins at the replication fork in E. coli Rep protein (helicase) 3’ 5’ pol III 5’ 3’ G Primasome DNA ligase C B Single-strand binding protein (SSB) DNA gyrase - this is a topoisomerase II, which breaks and reseals the DNA to introduce negative supercoils ahead of the fork pol III pol I

Components of the replication apparatus dna. A Primasome dna. B dna. C dna. G

Components of the replication apparatus dna. A Primasome dna. B dna. C dna. G DNA gyrase Rep protein SSB DNA pol III DNA pol I DNA ligase binds to origin DNA sequence helicase (unwinds DNA at origin) binds dna. B primase (synthesizes RNA primer) introduces negative supercoils ahead of the replication fork helicase (unwinds DNA at fork) binds to single-stranded DNA primary replicating polymerase removes primer and fills gap seals gap by forming 3’, 5’-phosphodiester bond

Properties of DNA polymerases of E. coli_ Polymerization: 5’ to 3’ Proofreading exonuclease: 3’

Properties of DNA polymerases of E. coli_ Polymerization: 5’ to 3’ Proofreading exonuclease: 3’ to 5’ Repair exonuclease: 5’ to 3’ pol III (core) yes yes no no DNA polymerase III is the main replicating enzyme DNA polymerase I has a role in replication to fill gaps and excise primers on the lagging strand, and it is also a repair enzyme • all DNA polymerases require a primer with a free 3’ OH group • all DNA polymerases catalyze chain growth in a 5’ to 3’ direction • some DNA polymerases have a 3’ to 5’ proofreading activity

Properties of DNA polymerases a DNA polymerases of humans b g d Location nucl

Properties of DNA polymerases a DNA polymerases of humans b g d Location nucl mito nucl Replication yes no yes Repair no yes Functions 5’ to 3’ polymerase yes yes 3’ to 5’ exonuclease no no yes 5’ to 3’ exonuclease 1 no no Primase yes no no no Associates with PCNA 2 no no no yes Processivity low high Strand synthesis lagging* repair both leading* * see notes below 1 activity present in associated proteins 2 Proliferating Cell Nuclear Antigen – “sliding clamp” 3 involved in transcription-linked DNA repair e nucl yes 3 yes no no yes lagging*

Proteins at the replication fork in humans helicase leading strand PCNA pol d 5’

Proteins at the replication fork in humans helicase leading strand PCNA pol d 5’ 3’ 3’ 5’ DNA ligase SSB topoisomerases I and II 5’ to 3’ exo associated with the complex pol e pol a (or pol d) primase activity lagging strand associated with pol a

Mutation Types and rates of mutation Type Genome mutation Mechanism chromosome missegregation (e. g.

Mutation Types and rates of mutation Type Genome mutation Mechanism chromosome missegregation (e. g. , aneuploidy) Frequency____ 10 -2 per cell division Chromosome mutation chromosome rearrangement (e. g. , translocation) 6 X 10 -4 per cell division Gene mutation base pair mutation (e. g. , point mutation, or small deletion or insertion 10 -10 per base pair per cell division or 10 -5 - 10 -6 per locus per generation

Mutation rates* of selected genes Gene Achondroplasia Aniridia Duchenne muscular dystrophy Hemophilia A Hemophilia

Mutation rates* of selected genes Gene Achondroplasia Aniridia Duchenne muscular dystrophy Hemophilia A Hemophilia B Neurofibromatosis -1 Polycystic kidney disease Retinoblastoma New mutations per 10 6 gametes 6 2. 5 43 32 2 44 60 5 to 40 to 5 to 105 to 57 to 3 to 100 to 12 *mutation rates (mutations / locus / generation) can vary from 10 -4 to 10 -7 depending on gene size and whethere are “hot spots” for mutation (the frequency at most loci is 10 -5 to 10 -6).

Polymorphisms exist in the genome • the number of existing polymorphisms is ~1 per

Polymorphisms exist in the genome • the number of existing polymorphisms is ~1 per 500 bp • there are ~5. 8 million differences per haploid genome • polymorphisms were caused by mutations New germline mutations • each sperm contains ~100 new mutations • a normal ejaculate has ~100 million sperm • 100 X 100 million = 10 billion new mutations • ~1 in 10 sperm carries a new deleterious mutation • at a rate of production of ~8 X 107 sperm per day, a male will produce a sperm with a new mutation in the Duchenne muscular dystrophy gene approximately every 10 seconds.

Types of base pair mutations normal sequence CATTCACCTGTACCA GTAAGTGGACATGGT transition (T-A to C-G) CATCCACCTGTACCA

Types of base pair mutations normal sequence CATTCACCTGTACCA GTAAGTGGACATGGT transition (T-A to C-G) CATCCACCTGTACCA GTAGGTGGACATGGT transversion (T-A to G-C) CATGCACCTGTACCA GTACGTGGACATGGT base pair substitutions transition: pyrimidine to pyrimidine transversion: pyrimidine to purine deletion CATCACCTGTACCA GTAGTGGACATGGT insertion CATGTCACCTGTACCA GTACAGTGGACATGGT deletions and insertions can involve one or more base pairs

Spontaneous mutations can be caused by tautomers Tautomeric forms of the DNA bases Adenine

Spontaneous mutations can be caused by tautomers Tautomeric forms of the DNA bases Adenine Cytosine AMINO IMINO

Tautomeric forms of the DNA bases Guanine Thymine KETO ENOL

Tautomeric forms of the DNA bases Guanine Thymine KETO ENOL

Mutation caused by tautomer of cytosine Cytosine Normal tautomeric form Guanine Cytosine Rare imino

Mutation caused by tautomer of cytosine Cytosine Normal tautomeric form Guanine Cytosine Rare imino tautomeric form Adenine • cytosine mispairs with adenine resulting in a transition mutation

Mutation is perpetuated by replication C G and C G • replication of C-G

Mutation is perpetuated by replication C G and C G • replication of C-G should give daughter strands each with C-G C A and C G • tautomer formation C during replication will result in mispairing and insertion of an improper A in one of the daughter strands C A T A • which could result in a C-G to T-A transition mutation in the next round of replication, or if improperly repaired

Chemical mutagens Deamination by nitrous acid

Chemical mutagens Deamination by nitrous acid

Derivation by hydroxylamine Alkylation by dimethyl sulfate causes depurination The formation of a quarternary

Derivation by hydroxylamine Alkylation by dimethyl sulfate causes depurination The formation of a quarternary nitrogen destabilizes the deoxyriboside bond and the base is released from deoxyribose

Attack by oxygen radicals

Attack by oxygen radicals

Thymine dimer formation by UV light

Thymine dimer formation by UV light

Summary of DNA lesions Missing base Acid and heat depurination (~104 purines per day

Summary of DNA lesions Missing base Acid and heat depurination (~104 purines per day per cell in humans) Altered base Ionizing radiation; alkylating agents Incorrect base Spontaneous deaminations cytosine to uracil adenine to hypoxanthine Deletion-insertion Intercalating reagents (acridines) Dimer formation UV irradiation Strand breaks Ionizing radiation; chemicals (bleomycin) Interstrand cross-links Psoralen derivatives; mitomycin C (Tautomer formation Spontaneous and transient)

Mechanisms of Repair • Mutations that occur during DNA replication are repaired when possible

Mechanisms of Repair • Mutations that occur during DNA replication are repaired when possible by proofreading by the DNA polymerases • Mutations that are not repaired by proofreading are repaired by mismatched (post-replication) repair followed by excision repair • Mutations that occur spontaneously any time are repaired by excision repair (base excision or nucleotide excision)

Mismatched (post-replication) repair • the parental DNA strands are methylated on certain adenine bases

Mismatched (post-replication) repair • the parental DNA strands are methylated on certain adenine bases CH 3 5’ 3’ CH 3 • the mutations are repaired by excision repair mechanisms • after repair, the newly replicated strand is methylated CH 3 • mutations on the newly replicated strand are identified by scanning for mismatches prior to methylation of the newly replicated DNA CH 3

Excision repair (base or nucleotide) deamination ATGCUGCATTGA TACGGCGTAACT uracil DNA glycosylase ATGC GCATTGA TACGGCGTAACT

Excision repair (base or nucleotide) deamination ATGCUGCATTGA TACGGCGTAACT uracil DNA glycosylase ATGC GCATTGA TACGGCGTAACT repair nucleases thymine dimer ATGCUGCATTGATAG TACGGCGTAACTATC excinuclease AT GCATTGA TACGGCGTAACT AT (~30 nucleotides) AG TACGGCGTAACTATC ATGCCGCATTGA TACGGCGTAACT ATGCCGCATTGATAG TACGGCGTAACTATC DNA polymerase b DNA ligase ATGCCGCATTGA TACGGCGTAACT ATGCCGCATTGATAG TACGGCGTAACTATC Base excision repair Nucleotide excision repair

Deamination of cytosine can be repaired Deamination of 5 -methylcytosine cannot be repaired More

Deamination of cytosine can be repaired Deamination of 5 -methylcytosine cannot be repaired More than 30% of all single base changes that have been detected as a cause of genetic disease have occurred at 5’-m. CG-3’ sites

Defects in DNA repair or replication • Xeroderma pigmentosum • Ataxia telangiectasia • Fanconi

Defects in DNA repair or replication • Xeroderma pigmentosum • Ataxia telangiectasia • Fanconi anemia • Bloom syndrome 100 human • Cockayne syndrome elephant Life span cow 10 hamster rat mouse shrew Correlation between DNA repair activity in fibroblast cells from various mammalian species and the life span of the organism 1 DNA repair activity

Defects in DNA repair or replication All are associated with a high frequency of

Defects in DNA repair or replication All are associated with a high frequency of chromosome and gene (base pair) mutations; most are also associated with a predisposition to cancer, particularly leukemia • Xeroderma pigmentosum • caused by mutations in genes involved in nucleotide excision repair • associated with a 2000 -fold increase of sunlight-induced skin cancer and with other types of cancer such as melanoma • Ataxia telangiectasia • caused by gene that detects DNA damage • increased risk of X-ray • associated with increased breast cancer in carriers • Fanconi anemia • increased risk of X-ray • sensitivity to sunlight • Bloom syndrome • caused by mutations in a a DNA helicase gene • increased risk of X-ray • sensitivity to sunlight • Cockayne syndrome • caused by a defect in transcription-linked DNA repair • sensitivity to sunlight • Werner’s syndrome • caused by mutations in a DNA helicase gene • premature aging