2 DNA Replication

Hydrogen bonding

• H bonding is most critical for DNA recognition by proteins

• occurs between amino acids side chains & the DNA bases & backbone

α−helical regions (that can fit into the major groove of B-form DNA)

α-helices, major groove

MULTIPLE DNA-binding domains in a protein

a group of proteins that binds DNA at origins of replication & helps initiate replication in YEAST (a eukaryote*)

initiation sites for DNA replication in a single mammalian chromosome

a model system used to outline the basic features of human DNA replication

the viral protein T-antigen (T-ag) is not found in humans & is the one exception - otherwise the same human proteins are used to synthesize SV40 & human DNA

the identification of proteins involved in human DNA replication

recognizes and binds to the SV40 origin & then serves as a DNA helicase (finds the origin → melts it)

analagous to yeast ORC (origin recognition complex)

enzymes that use the energy of nucleoside triphosphate hydrolysis (eg. ATP) to break hydrogen bonds that hold DNA strands together in the double helix

• what nature uses instead of heat & NaOH to unwind duplex DNA

the human DNA Helicase (it unzips dsDNA, resulting in the formation of single strands)

1. T-ag

2. Human SSB (RPA)

3. Topoisomerase I

protein that binds to ssDNA ONLY & stabilizes it (prevents re-annealing)

is required for DNA replication

human SSBs are known as RPAs (Replication Protein A)

removes positive supercoiling in front of a DNA replication fork (changes topology)

does this by breaking a single DNA strand, allowing the DNA structure to relax, then covalently re-connecting the DNA strands where they were originally cut

DNA Helicase

Bloom’s is associated with a high risk of developing malignancy (Leukemia)

premature aging, dwarfism, cataracts, scleroderma like skin changes, alopecia, caused by a mutation in a HELICASE gene

5' → 3'

associates with DNA polymerase α & resulting complex synthesizes a short RNA strand that serves as a primer for other DNA polymerases

works with Primase to make the primers required to initiate DNA-synthesis by pol ε (leading) & pol δ (lagging)

synthesizes the leading strand during DNA replication

is a HIGH FIDELITY polymerase with 3' - 5' exonuclease proofreading activity

is PCNA DEpendent

synthesizes the lagging strand during DNA replication

is a high fidelity HIGH FIDELITY polymerase with 3' - 5' exonuclease activity

requires PCNA to remain attached to the DNA template

forms a clamp around a DNA polymerases ε & δ & prevents them from dissociating from the DNA template strand

clamp loader (clamp = PCNA)

once the clamp is in place then polymerases can be recruited for chain elongation

covalently joins DNA ends & Okazaki fragments to one another

synthesizes mitochondrial DNA

participates in excision repair; maintains genome integrity

(DON’T confuse with RNA polymerase β which synthesizes mRNA)

a nucleotide analog that inhibits Herpes simplex viruses polymerase

• it doesn’t have a 3’ -OH so it’ll be incorporated but transcript elongation can’t occur

inhibits HIV reverse transcriptase

• normally, the 3’ -OH bonds to the next nucleotide at it’s 5’ phosphate → chain growth

• AZT has a 3’ N3 that prevents phosphodiester bond formation → chain terminates

1. it’s TOXIC: inhibits other polymerases (eg. mitochondrial DNA pol γ)

2. eventually mutant forms of reverse transcriptase arise that have a lower affinity for the drug (have a higher Km, takes more substrate to saturate enzyme)

Mitosis (M) & Interphase

includes G1, S, & G2

part of the cell cycle where it’s not in mitosis

chromosome condensation takes place

1. G1 (growth; includes G1)

2. S (DNA synthesis)

3. G2 (more growth)

4. M (Mitosis, which itself is divided into several stages)

G0 (can occur within G1)

S phase (S for synthesis)

enzymes that regulates progress through the cell cycle by adding phosphate groups to other molecules

made up of a kinase and a cyclin

changes in the kinase & cyclin components (A-E) drive the cell from 1 stage of the cell cycle to another

a tumor suppressor protein active at the G1/S checkpoint that prevents cells with DNA damage from replicating (before S phase, where replication [synthesis] occurs)



• is mutated in over 50% of cancers

• makes sure damaged DNA isn’t a substrate for replication

condition caused by mutant inherited versions of the p53 tumor suppressor gene

results in tumors at multiple sites

programmed death that cells in which the genome is so damaged that it can’t be repaired may undergo

detects replication errors, eg. A — C = a mismatch

uses parental template strand’s adenine methylation to target other strand for base repair

• Mut H
• Mut L
• Mut S

Mut S recognizes mismatched base pairs

catalyzes the incision of the section of the strand containing the mismatched base pairs

function unknown; it’s thought to provide a protein-protein interface between Mut S & Mut H

hMSH (Human Mut S Homolouge)

hMLH (Human Mut L Homolouge)

mutations in hMSH & hMLH genes are correlated with hereditary non-polyposis colon cancer (HNPCC) + a predisposition to other cancers

• defects in this system may be related to trinucleotide repeat expansions

1. Fragile-X syndrome (CGG, FMR-1)

2. Spinobulbar Muscular Atrophy (CAG, androgen receptor)

3. Myotonic Dystrophy (CTG, myotonin kinase)

4. Huntington's Disease (CAG, huntington)

* these disorders frequently involve the brain

• • the greater # of repeats → earlier the disease onset
• • replication through these repeats can be a problem
• • IF the mismatch repair system is functional it’ll fix errors
• • when the mismatch repair system is nonfunctioning, disease results
• • an important function of the mismatch repair system is to NOT let trinucleotide repeats expand* (not formally proven)

moderate to severe mental retardation stemming from failure of normal neural development

expansion to greater than 200 repeats in the FMR gene on Chromosome 11

multisystem (eg. muscle weakness, endocrine dysfunction)

dramatic expansions of repeats: ~1000

neurodegenerative disorder with inverse correlation between number of repeats & age of onset :(

• 1. Incision
• 2. Excision
• 3. Resynthesis
• 4. Ligation
• (modeled in prokaryotes)
• 

covalent bond formation between adjacent thymines that occurs when DNA is exposed to ultraviolet light (or certain chemical agents, cigarette smoking)

• resulting pyrimidine dimers don’t fit into a double helix so replication & gene expression are blocked until lesions are removed

• considered a bulky addict

Excision Repair

makes sense because strand stabilization & strand resynthesis needs to happen with excision repair as well

skin cancer correlated with defective excision repair

as a result UV-induced DNA damage cannot be effectively repaired

• XP is the classic example of why we know excision repairs is important; if you can’t remove thymine dimers the end result is cancer

Uracil

this is potentially mutagenic as it can lead to mismatched base pairs

removes Uracil (that’s arisen because of cytosine deamination) to create an ?AP site? (apurinic or apyrimidinic)

there’s then an endonuclease that CUTS the strand & provides a primer that allows for resynthesis by DNA pol

• Nitrous Acid (HNO2)
• 
• Deamination of Adenine → Hypoxanthine (acts like guanine (G) in DNA)
• Deamination of Cytosine → Uracil

the N7 of guanine



this weakens N-glycosidic linkages in DNA & leads to a loss of Guanine from the DNA

polycyclic rings that have the ability to insert themselves between bases of DNA causing physical distortion of the DNA & ultimately leading to the addition or loss of base pair(s)

drugs can be intercalators

protein complex responsible for the repair of pyrimidine dimers in bacteria