03: 16 Dna Replication/ Gene Mutations/ Dna Repair (Kc)

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• Semiconservative Replication

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- 2 parental strands build new strands that are complementary to the original strands, so that each has one strand that is parental and one that is new

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o Note that DNA replication happens in what phase?

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“S” phase.

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• Prokaryotic DNA Replication

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(All-or-Nothing)

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What are the steps of DNA replication?

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1) Identify the sequence of origin (start replication)
2) Separate (helicase) and unwind (topoisomerase) DNA strands
3) Keep strands apart with single-strand binding proteins (SSBs)
4) Make a RNA primer (primase)
5) Create the new DNA strand (DNA polymerase)
6) Remove primer and fill gap with deoxyribonucleotides
7) Bind any holes/nicks in phosphate backbone (ligase)

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Prokaryotic DNA Replication

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All-or-Nothing

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DnaA binds sequence at

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at oriC site (w/ GATC/CTAG repeats)

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“A” (Adenine) sites methylated by

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Dam methylase

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Replication forks move in

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opposite directions

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Helicase (DnaB)

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breaks hydrogen bonds between parental strands

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Topoisomerase (DNA gyrase)

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untwists parental DNA (breaks one strand)

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Single-stranded binding proteins (SSBs)-

Back 11

bind to ssDNA to prevent re-annealing

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o DNA Polymerases

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“workers” of replication

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Pol I

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Fill Gap after removal of RNA primase, DNA repair (5’ to 3’ and 3’ to 5’)
• Assists in removal of primer

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Pol II

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DNA repair (3’ to 5’)

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Pol III

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Replication (5’ to 3’) and Proofreading (3’ to 5’)

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o Primase (DnaG)

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8-10 nucleotide RNA primer on ssDNA.

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Note: Replication is always 5’ to 3’, meaning one strand is continuous (__), while the other is discontinuous (lagging strand), and requires DnaG many times along its length, forming Okazaki fragments

Back 17

Note: Replication is always 5’ to 3’, meaning one strand is continuous (leading strand), while the other is discontinuous (lagging strand), and requires DnaG many times along its length, forming Okazaki fragments

Front 18

Note: Replication is always 5’ to 3’, meaning one strand is continuous (leading strand), while the other is discontinuous (__), and requires DnaG many times along its length, forming Okazaki fragments

Back 18

Note: Replication is always 5’ to 3’, meaning one strand is continuous (leading strand), while the other is discontinuous (lagging strand), and requires DnaG many times along its length, forming Okazaki fragments

Front 19

Note: Replication is always 5’ to 3’, meaning one strand is continuous (leading strand), while the other is discontinuous (lagging strand), and requires DnaG many times along its length, forming _ _.

Back 19

Note: Replication is always 5’ to 3’, meaning one strand is continuous (leading strand), while the other is discontinuous (lagging strand), and requires DnaG many times along its length, forming Okazaki fragments

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o Ligase seals phosphate backbone between 2 fragments, forming

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phosphodiester bond

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o Type 2 Topoisomerase

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Unlinks two new circular chromosomes

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Eukaryote DNA Replication (note - changing topics. Skip card)

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skip

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Origin Recognition Complex (ORC)

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binds at points of origin, while the minichromosome maintenance proteins (MCM) binds to DNA to open strands

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Once open, helicase, topoisomerase, and SSBs (RPA) can __

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bind

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o Sliding Clamp (PCNA)

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wraps around DNA and travels along strand with DNA Polymerase, increasing processivity.

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o Primase and Pol alpha

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add 10 nucleotide RNA primer and 20 nucleotide dNTPs
Primer removed by flag endonuclease 1 (FEN1) and RNase H.

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o Pol Epsilon

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continues replication on leading strand

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o Pol Delta

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continues replication on lagging strand (Okazaki fragments) and fills in missing DNA sequence in primer region

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o Ligase joins

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phosphate backbone

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Replication of Telemeres: Problem on lagging strand, resulting in

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a shorter strand.

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o Telomerase lengthens

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3’ overhand with primer that recognizes TTAGGG sequence

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Telomerase also acts as a

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polymerase

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• Point Mutations

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single nucleotide change

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o Missense Mutation

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change in amino acid sequence

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o Nonsense Mutation

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change to a stop codon

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o Insertion

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Addition of a nucleotide (changing reading frame)

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o Deletion

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Deletion of a nucleotide (changing reading frame)

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DNA Damage (Other Factors Aside from Error Rate)

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Chemicals, UV/Radiation, Other Forms

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o Deamination

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- spontaneously removal of amine group from cytosine, converting it to uracil.

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 Adenine converts to

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hypoxanthine

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 Guanine converts to

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xanthine

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 Thymine has

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no deamination

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o Depurination

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Cleavage of glycoyl bond between purine and the deoxyribose
(i.e.- the strand that contains the purine has a deletion introduced upon replication, while the other strand remains normal when replicated)

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• UV/Radiation

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o Thymine dimers (cyclobutane ring)- causes abnormal indentation in the structure
o Benzo[a]pyrene- Compound found in cigarette smoke, which distorts DNA helix and allows dimers to form between bases.

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• Other Forms
o Oxidation of DNA bases and Alkylation

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adding methyl and alkyl group

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• Base Excision Repair

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Glycosylase marks and removes base, endonuclease cuts strand, polymerase synthesizes it and DNA ligase fills in phosphate backbone

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• Nucleotide Excision Repair

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same as above, but detects distortions in DNA helix.

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Xeroderma pigmentosum

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Defect: defects in nucleotide-excision repair
Symptoms: freckle like spots on skin, sensitivity to sunlight, predisposition to skin cancer.

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Cockayne syndrome

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Defect: defects in nucleotide-excision repair
Symptoms: dwarfism, sensitivity to sunlight, premature aging

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Trichothiodystrophy

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Defect: defects in nucleotide-excision repair
Symptoms: brittle hair, skin abnormalities

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Hereditary nonpolyposis colon cancer

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Defect: defects in mismatch repair
Symptoms: predisposition to colon cancer

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Fanconi anemia

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Defect: Possibly defects in the repair of interstrand cross-links
Symptoms: increased skin pigmentation, abnormalities of skeleton, heart, and kidneys, predisposition to leukemia

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Ataxia telangiectasia

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Defect: Defects in DNA damage detection and response
Symptoms: Defective muscle coordination, dilation of blood vessels in skin and eyes,… predisposition to cancer

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Li-fraumeni syndrome

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Defect: Defects in DNA damage response
Symptoms: Predisposition to cancer in many different tissues

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• Homologous Recombination

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repairs double-strand breaks
o Nuclease generates single-stranded ends, pairs with complementary DNA strand as the invading strand, forming a branch point and elongating by DNA polymerase

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• Nonhomologous DNA Repair

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Simple way to repair double strand breaks, by cutting out the error and ligating the strands back together, at the cost of a deletion in the DNA.

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o Normally not a problem in humans because

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we have so much non-coding DNA.