Protein-DNA interactions are importantly made possibly by what?
• H bonding is most critical for DNA recognition by proteins
• occurs between amino acids side chains & the DNA bases & backbone
What regions do proteins often contain that help interaction with DNA?
α−helical regions (that can fit into the major groove of B-form DNA)
Many proteins contain ____ (structural motif) that can fit in the ____ (location) of DNA to play a role in site specific binding
α-helices, major groove
What is frequently required for site-specific recognition?
MULTIPLE DNA-binding domains in a protein
Origin Recognition Complex (ORC)
a group of proteins that binds DNA at origins of replication & helps initiate replication in YEAST (a eukaryote*)
Origins of Replication
initiation sites for DNA replication in a single mammalian chromosome
Simian Virus 40 (SV40) Model System
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
What did studying the replication of SV40 DNA in extracts prepared from human cell lines lead to?
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
MCM (Minichromosome Maintenance Complex)
the human DNA Helicase (it unzips dsDNA, resulting in the formation of single strands)
Name 3 replication initiation factors:
2. Human SSB (RPA)
3. Topoisomerase I
SSB (Single-stranded Binding Protein)
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
What is deficient in children with Bloom’s Syndrome?
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
In which direction do all DNA & RNA polymerases synthesizing new strands of DNA read their templates?
5' → 3'
associates with DNA polymerase α & resulting complex synthesizes a short RNA strand that serves as a primer for other DNA polymerases
DNA Polymerase α
works with Primase to make the primers required to initiate DNA-synthesis by pol ε (leading) & pol δ (lagging)
DNA Polymerase ε
synthesizes the leading strand during DNA replication
is a HIGH FIDELITY polymerase with 3' - 5' exonuclease proofreading activity
is PCNA DEpendent
DNA Polymerase δ
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
PCNA (Proliferating Cell Nuclear Antigen)
forms a clamp around a DNA polymerases ε & δ & prevents them from dissociating from the DNA template strand
RFC (Replication Factor C)
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
DNA Polymerase γ
synthesizes mitochondrial DNA
DNA Polymerase β
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
Zidovudine (ZDV)/Azidothymine (AZT)
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
Why is AZT not 100% effective?
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)
What 2 phases can the cell cycle be divided into?
Mitosis (M) & Interphase
includes G1, S, & G2
part of the cell cycle where it’s not in mitosis
chromosome condensation takes place
What are the four stages of the cell cycle?
1. G1 (growth; includes G1)
2. S (DNA synthesis)
3. G2 (more growth)
4. M (Mitosis, which itself is divided into several stages)
What is the quiescent phase of the cell cycle?
G0 (can occur within G1)
During what phase does DNA replication take place?
S phase (S for synthesis)
Cyclin-dependent Protein Kinase (Cdk)
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
Prokaryotic Mismatch Repair System
detects replication errors, eg. A — C = a mismatch
uses parental template strand’s adenine methylation to target other strand for base repair
What are the 3 proteins used in prokaryotic mismatch repair?
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
What are the 2 human proteins involved in mismatch repair?
hMSH (Human Mut S Homolouge)
hMLH (Human Mut L Homolouge)
What are mutations in human mismatch repair genes correlated with?
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
Diseases Associated with Trinucleotide Repeats (4):
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
Mismatch Repair System, Trinucleotide Repeats & Disease
- • 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)
Fragile X Syndrome
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 :(
4 Steps of Excision Repair (in E.coli)
- 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
Proteins like PCNA & SSB are required for what process in addition to replication?
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
What base can Cytosine spontaneously deaminate to form?
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
What’s a chemical mutagen that can cause deamination?
- Nitrous Acid (HNO2)
- Deamination of Adenine → Hypoxanthine (acts like guanine (G) in DNA)
- Deamination of Cytosine → Uracil
What is the major site of alkylation on DNA bases?
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