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Flashcards in DNA Replication Deck (18):
1

Structure of DNA - sugars

– ribose or deoxyribose
 C1’ – nitrogenous base attaches via N-glycosidic bond
 C2’ – H (DNA) or OH (RNA)
• lack of oxygen in DNA makes DNA more stable
 C3’ – OH in both
 C5’ – has 1-3 phosphate groups attached via ester linkage

2

structure of DNA - nitrogenous bases

 Pyrimidines – 1 ring – cytosine, thymine, and uracil (RNA)
• Thymine has a methyl group whereas uracil does not
• Cytosines can have a methyl group added when next to guanine
o Modification that changes how proteins interact with DNA, influences its structure indirectly, and plays important role in regulation of gene expression
 Purines – 2 rings – adenine and guanine
 Deamination – spontaneous removal of an amine group (-NH2) from a nucleotide
• Common type of DNA damage
• Deaminated cytosine = uracil  easily detected
• Deaminated adenine = hypoxanthine  easily detected
• Deaminated methyl-cytosine = thymine  NOT easily detected and repaired

3

structure of DNA - phosphate groups

 1-3 phosphate groups (alpha, beta, gamma) – alpha is closest to the sugar and the only one incorporated in nucleic acids; negatively charged
• Hydrolysis of other to phosphate groups provide energy

4

Nucleotide

= a nucleoside triphosphate
nucleoside = sugar + nitrogenous base

5

DNA Helix Structure

o Nucleotides joined by phosphodiester bonds
o Polar
 5’ PO4
 3’ OH
o H bonds between nitrogenous bases
 A-T base pairs have 2 H bonds
 G-C base pairs have 3 H bonds
o Purine always base paired with pyrmidine
o Anti-parallel strands that coil around an axis of symmetry
o Alpha-helix
 Bases lie perpendicular to axis of symmetry
 ~10 bp per turn
 Major groove = between turns
 Minor groove = between strands
o Proteins interact with DNA via the major and minor grooves
o Complimentary base pairs and antiparallel structure allows both strands to be used as templates for new DNA synthesis and allows for DNA repair

6

packaging DNA into the nucleus

o Chromatin = DNA + proteins
• Euchromatin – less condensed; transcriptionally active
• Heterochromatin – more condensed; inactive
 Mitosis – chromatin becomes very condensed and chromosomes are visible
o 3 billion base-pairs of DNA compacted into nucleus 10,000:1
o Nucleoli – sites of ribosome synthesis
o Nucleosome – “beads on a string”
o Nucleofilaments – 30nm fibers of packed nucleosomes in a “2 start helix” consisting of 2 strands of nucleosomes stacked like coins
 Anchored via scaffolding proteins to form chromosomes

7

chromatin structure and DNA replication and transcription

o Proteins involved in replication and transcription must get around histone proteins
o Histones have positively charged lysine amino acids that interact with negatively charged phosphate groups of DNA
o Histone acetyl transferases (HATs) – add acetyl groups
 Favors formation of euchromatin (active)
o Histone deacetylases (HDACs) – remove acetyl groups
 Favors formation of heterochromatin (inactive)
o Other types of post-translational modifications – phosphorylation, methylation, ubiquitination

8

DNA replication - Cell cycle

 Checkpoints that regulate cell cycle progression
• G1 – are chromosomes intact after mitosis
• G2 – did all the DNA get replicated once and only once
 DNA damage results in cell cycle being halted and initiation of repair pathways
• If DNA damage cannot be repaired the cell undergoes apoptosis
 S phase – DNA synthesis phase ~6-8 hours
• Regions of genome that are actively transcribed are replicated in early S phase
• Regions of genome that are untranscribed are replicated in late S phase
 Licensing – process that ensures that all regions of DNA are replicated once and only once per cycle

9

DNA replication features

 Semi-conservative – each of 2 daughter strands contain one strand of parental DNA and one strand of newly synthesized DNA
 Multiple origins of replication
• Bidirectional – fast and efficient – 5’  3’
• Replication forks
• Replication bubble – region of unwound DNA

10

Initiation of DNA replication

 Each origin of replication is marked/licensed in late G1; once replicated in S phase the license is removed; ensures same area of DNA is not copied twice
 Origin Recognition Complex (ORC) assembles at origin throughout cell cycle
• Cannot initiate replication on its own
 Cdc6 and Cdt1 bind to ORC in G1 and recruits Mcm to form pre-replicative complex (preRC) – origin is now licensed
 S-Cdk phosophorylates Cdc6, Cdt1 and Mcm causing the disassembly of preRC and initiation of transcription
 Mcm has helicase activity that form replication bubble; it migrates with the replication fork

11

movementof the replciation fork requires several proteins

 Helix must be unwound in order to separate the strands; supercoiling that results has to be relieved
 Helicase enzymes (including Mcm) move along one strand of DNA and change conformation to bind to double strand; using ATP they separate the two strands and return to original conformation
 Single strand binding proteins (including RPA) bind to single stranded regions and prevent the two strands from re-annealing
 Topoisomerases (type 1) – make reversible nicks in DNA ahead of replication forks, pass unbroken strands through the gap, and then reseal the gap
 Topoisomerases (type 2) – make double strand breaks in the DNA, allow uncoiling, and then re-ligate
• Clinical Scenario – Topoisomerases – targets for chemotherapy for cancer
• Clinical Scenario – Base analogs substitute for normal bases and inhibit DNA replication (often used for anti-virals or to treat cancer)

12

DNA synthesis by DNA polymerase

 DNA polymerase creates a phosphodiester bond between 5’ PO4 of incoming nucleotide and 3’ OH of previous nucleotide
 DNA syntheses occurs in 5’ to 3’ direction but moves along the template 3’ to 5’
 First 3’ OH is provided by an RNA primer made by primase (an RNA polymerase)
 DNA polymerase has 3’5’ exonuclease activity which allows it to proofread by excising the last nucleotide added if it doesn’t base pair correctly with template
 Leading strand is copied continuously from origin of replication in same direction as replication fork
 Lagging strand is copied discontinuously in small fragments beginning at replication fork and moving toward origin; fragments are called Okasaki fragments
 RNA primers are removed by RNA hydrolases (RNases)
 DNA ligase uses ATP to make the final phosphodiester bond to seal nicks and join the fragments together
 PCNA (proliferating cell nuclear antigen) is a sliding clamp that increases the processivity of DNA polymerase; also involved in DNA repair, chromatin remodeling, and cell cycle regulation
 Multiple (atleast 14) DNA polymerases

13

maintenance of epigenetic signals

o Epigenetics – study of heritable changes in gene expression that occur without changes in the primary DNA sequence
o Epigenetic Signals – histone modifications and DNA methylation
 Patterns are maintained during DNA replication
 Influence gene expression through effects on chromatin structure by regulating accessibility of the DNA to transcription factors
 Most forms of epigenetic inheritance are not passed to the next generation because they are erased during the production of germ cells

14

patterns of DNA methylation

 On cytosines next to guanines on the same strand
 Associated with heritable gene inactivation (inhibits transcription)
 Important for development, X chromosome inactivation, imprinting
 Necessary to maintain chromosomal stability by keeping repetitive sequences in non-coding region in a repressed state
 Many CG sequences have mutated to TG via deamination throughout evolution
 CG islands (rich CG areas), often at 5’ end of ‘housekeeping genese’ that are constitutively expressed, are normally NOT methylated
 Since methylation in 5’ regulatory regions of genes suppresses gene expression, patterns of DNA methylation represent a type of epigenetic information that must be passed on to each of two daughter cells
 Same patterns of methylation are maintained during DNA replication by DNA maintenance methylase that recognizes hemi-methylated sites and methylates the other, newly synthesized strand

15

histone code

 Histone tails often modified by acetylation, methylation, phosphorylation, ubiquitination
 Modification = epigenetic signal that helps regulate gene expression
 Histones are distributed during replication so that they leave gaps
 New histone proteins bind the gaps and are modified by reader-writer remodeling complexes to match the ones already bound

16

problem of end replication

– because of removal of the RNA primer from the end of the lagging strand, the 3’ end of the parental DNA cannot be replicated completely
 Telomeres protect the ends of chromosomes
• Many copies of 6 base pair DNA sequence, plus proteins that bind
• 3’ overhang folds back and provides structure for proteins to bind
• Functions to protect ends of chromosomes from degradation and fusion
• Functions to distinguish ends of intact chromosomes from broken ones
• Telomeric DNA is lost with every replication of DNA, fewer proteins can bind; and risk increases that telomeres will not be able to protect ends of chromosomes
• Loss of telomeric DNA triggers cell cycle checkpoint mechanisms and causes the cell to stop growing permanently (cells are said to be senescent)

17

telomerase and mechanism of activity

• Length of telomeres is maintained in germ line cells and some stem cells by telomerase, a ribonucleoprotein that carries its own RNA template complimentary to the G-rich strand of the telomere
o Enzyme is NOT detectable in normal somatic cells
o Found in germline cells, stem cells and >85% of cancer cells
• Expression associated with immortalization of cell lines
• Uses RNA template to extend 3’ end of parental strand (reverse transcriptase)
• Experimental cancer vaccines are being developed that target telomerase as tumor-specific antigen

18

mitochondrial DNA

o Some mitochondrial proteins are transcribed and translated in the mitochondria but most are transcribed in the nucleus and translated in the cytosol before the protein is imported to mitochondria
o Mitochondrial DNA is more economical than nuclear DNA
 Every base pair is used to make a functional product whereas nuclear genome has a lot of repetitive DNA
o Mitochondrial DNA has higher mutation rate than nuclear DNA (5-10 times)
 Partly due to its close proximity to sites of generation of oxygen radicals but also may be due to less efficient repair mechanisms
o Accumulated mutations in mitochondria DNA have been suggested to contributed to decreased efficiency of oxidative metabolism with increased age and overall aging process