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molecular bio


copy deck
3 components of nucleic acids
base (nitrogen), sugar (pentose), phosphate
Rings in a Purine
Rings in a Pyrimidine
Purine Nucleotides
Adenine and Guanine
Pyrimidine Nucleotides
Cytosine, Uracil and Thymine
What is the central dogma of molecular biology
DNA --> RNA --> Protein
What is the net charge on an ATP molecule
How do nucleotides bond to each other
Bonds in A-T (or A-U)
Bonds in C-G
What carbon is the glycositic linkage at in the ribose backbone?
What carbon is the site of phosphate addition in the ribose backbone?
What carbons establish polarity in the ribose backbone?
3' and 5'
What carbon is the phosphodiester bond at in the ribose backbone?
3' and 5'
What hyrdoxide group is different between ribose and deoxyribose
What is the difference between a nucleoside, nucleotide, and nucleic acid
Nucleoside=base+sugar, nucleotide=base+sugar+phosphate, nucleic acid=polymer of nucleotides
In what direction is the nucleotide sequence read?
5' -> 3'
What are the grooves in DNA?
Major and minor
What was an experiment that showed that DNA was the genetic material?
labeled proteins and DNA in a phage with radioactive labels, DNA correlated with virulence
What were the Griffith, Avery MacCleod and MacCarthy experiments?
Showed that genetic material from heat-killed bacteria could cause non-toxic bacteria to be toxic.
What is DNA supercoiling and what are the two forms it takes?
positive (overwound, unstable+left turns), negative (underwound, stable+right turns)
How can you untangle messy DNA
DNA treated with heat and base will do what?
Undergo strand separation
What do topoisomerases do?
Nick DNA
How do topoisomerases work as therapeutic agents
Ciproflaxin and novobiocin (antibiotics) inhibit bacterial gyrases, Etoposide and Doxorubicin affect eukaryotic type II topoisomerases in cancer treatment
What is the shape of the eukaryotic genome? Viruses? Bacteria
large condensed chromosomes, linear, and circular (supercoiled) for bacteria
How many chomosomes to humans have?
22 autosomes + XX or XY
What is chromatin?
Highly organized structure with histone proeins that bind and twist DNA around them to form nucleosomes
What is a histone?
a protein that forms the core of a nucleosome
What is a nucleosome?
A complex of 4 pairs of histones (H2A, H2B, H3, H4) referred to as the octamer, that DNA wraps around two times
What is a solenoid?
A 30nm chromatin fiber composed of 6 nucleosomes
What joins nucleosomes together
linker DNA of variable length and histone H1
How can histones be modified?
acetylation (activates), deacetylation (deactivates), methylation (activation and inactivation), phosphorylation (increases condensation), and ubiquitination
What is Cofin Lowry Syndrome
mutation in RSK (kinase that phosphorylates histones to further condensation of chromosome); mutation disrupts gene expression, leading to devel. Defects and mental retardation.
What are diseases that are involved in DNA methylation?
ICF syndrome, Rett syndrome
What is fragile-X chromosome and what causes it?
Most common heritable form of mental retardation-- trinucleotide expansion repeat in FRAX gene fragile site.
What is the centromere?
Non-coding region of attachment of p and q chormosome ends, site of spindle attachment during mitosis
What is the telomere?
non-coding region at each end of the chromosome, created by telomerase, prevents chromosome shortening in replication
What classes of sequences are there in the human genome?
Unique 1-10 copies per genome, middle repetetive (10-10,000) tRNA and rRNA genes, higly repetitive (>100K) non-coding regions
What is replication, transcription, translation?
DNA synthesis (DNA pol), RNA synthesis (RNA pol), protein synthesis (cellular ribosomes and tRNA)
What diseases are examples of a misregulation of DNA replication?
Cancer, viral disease
How can you experimentally differentiate between a DNA synthesis initiation protein and an elongation protein?
Use heat shock protein to stop synthesis; an initiation protein will stop replication after a lag time, an elongation protein will stop synthesis immediately
What direction is DNA sythesized?
How are nucleotides added to a growing DNA chain?
NTP covalently attaches to the 3' OH, and Ppi is released.
What do di-deoxynucleotides do?
Stop synthesis of DNA
The region of the genome serviced by one origin of replication
The complex of proteins that function at the replication fork
What is a theta structure?
A plasmid that has begun to replicate--it has a fork and it looks like an eye
What is primase?
an RNA polymerase, initiates replication by adding short complementary RNA primers to both strands of DNA
Okazaki fragment
Short segment of DNA synthesized between primers on the lagging strand
2 requirements for DNA replication
proteins, RNA synthesis
Four prokaryotic polymerases (not all)
Primase, DNA Pol I, DNA Pol II, DNA Pol III
DNA polymerase I
5' to 3' DNA synthesis, 3' to 5' proofreading, (okazaki fragments) 5' to 3' exonuclease replaces primers with DNA
DNA polymerase II
5' to 3' DNA synthesis, 3' to 5' proofreading
DNA polymerase III
5' to 3' DNA synthesis (major DNA polymerase), 3' to 5' proofreading
What are some features of the origin of replication?
9'mers and 13-mers bind DnaA, A-T rich to facilitate unwinding, protein binding sequences
What is DnaA
binds to 9-mers in eukaryotes to initiate synthesis
What do DnaB and DnaC do?
DnaB is a helicase, unwinds DNA after DnaC loads it on the DNA and lets go (ATP dependent)
What is DnaG?
Bacterial primase
What are SSBs?
Single-stranded binding proteins, protect ssDNA from degradation
What comprises the DNA Pol III holoenzyme?
Clamp loader, clamp (homodimer), core enzyme, DNA pol dimerizer
How is replication terminated?
tus proteins bind ter sites-- DNA Pol III and DnaB fall off, terminating replication
Why are topoisomerases important in replication
Because helicase would have to rotate the DNA 50 times/second to keep unravelling it
What are the eukaryotic DNA synthesis enzymes?
DNA pol alpha, DNA pol delta, DNA pol epsilon
DNA polymerase alpha
Primase, makes RNA primers+first few dNTPs, 5'-3' RNA and DNA synthesis
DNA polymerase delta
DNA elongation, 5'-3' DNA synthesis and 3'-5' proofreading
DNA polymerase epsilon
DNA elongation, exact function unknown
What is processivity?
The ability to synthesize a long stretch of DNA and not fall off
Process by which cell is induced to enter the S phase
pre-replication complex
Forms at origin of replication, necessary for licensing
Key proteins involved in licensing
ORC, Cdt, Cdc6/18, MCM proteins, Geminin
blocks licensing by (possibly) interacting with Cdt1, also disassembles replication machinery
Cdt1/Cdc6/18/MCMs complex
pre-replication complex, forms when Geminin is degraded and initiates replication
Eukaryotic helicase
Eukaryotic equivalent of DnaA
ORC, Dcc6/18, Cdt1
Eukaryotic clamp loader
Eukaryotic clamp
DNA pol dimerizer (eukaryotes)
Not known
Eukaryotic RNA removal
Ligase I
What is SV40
animal virus isolated from African Monkey, used as model for mammalian replication machinery
T antigen
key component of SV40 DNA replication machinery--acts as pre-replication complex
Single-stranded binding proteins, protect ssDNA from degradation
What does telomerase do?
prevents strand shortening because of lagging strand problems with synthesis
What end extends too far at the end of a chromosome?
End of DNA bound to itself by telomerase to cap chromosome
ss 3' loop invades the duplex and hybridizes
Telomerase activity
high in germ cells/embryonic cells; as differentiation increases, activity decreases
Abnormal telomerase activity
many cancer cells/immortalized cells reactivate telomerase
2 cell cycle proteins modified by viruses
p53 (temporarily prevents entry into S phase when DNA is damaged), Rb (binds and inhibits E2F, a TF that would otherwise induce expression of replication proteins)
Characteristics of SV40
causes tumors when injected into rodents, no sign of disease in humans, genome=ds circular DNA, model for eukaryotic replication
T antigen's effects in host cell
Binds p53, inactivating it; binds Rb, inactivating it; also acts as pre-replication complex
SV40 replication procedure
SV40 encodes LT, 2 LT complexes bind minimal origin, eukaryotic machinery engages replication (LT is helicase), topoisomerase separates+uncoils plasmids
Adenovirus activity (E1A and E1B)
E1A binds RB blocking its binding to E2F, E1B binds p53, inactivating it
Drugs that target replication
AZT, ddI and ddC, Acyclovir, Actinomycin, Acridine dyes, Daunorubicin
AZT mechanism of action
Azidothymidine, AKA Zidovudine, T analog lacking 3' OH, specific for HIV reverse transcriptase
ddI and ddC mechanism of action
Analogs that lack 3' OH--used to treat HIV
Acyclovir mechansim of action
G analog lacking 3' OH, specific for HSV and Herpes Varicella Zoster (chicken pox) DNA polymerase
Actinomycin, Acridine dyes, Daunorubicin mechanism of action
intercalating agents that incorporate into the DNA duplex and disrupt replication, transcription and recombination processes
Adenovirus genome
ds linear DNA, undergoes replication as circularized then linear DNA, contains 3' pTP
Rolling Circle replication
Herpes virus linear DNA ligates into a circle, replicates as a theta structure, then as a rolling circle.
What is a DNA polymorphism?
single or multi-base pair change in genetic code
What is the difference between a functional and a molecular polymorphism?
a functional polymorphism occurs within a gene and may affect its function; a molecular polymorphism (eg sickle cell anemia polymorphism) occurs close to a gene and may not affect its function-- it can often be used as a marker to identify the gene
What does a restriction enzyme do?
Recognizes and cleaves specific DNA "recognition" sequences
What is the difference between an endonuclease and an exonuclease?
endonucleases recogize dsDNA and cleave leaving blunt or a sticky ends; exonucleases recognize the ends of DNA strands and digest them--exonucleases are especially important in apoptosis
How do bacteria protect themselves from their own endonuclease activity?
By pairing restriction endonucleases with DNA modification enzymes (methylases)--methylated DNA will not be cut.
For the purposes of this course, what is a clone?
a segment of DNA of interest and its vector-- can be manipulated/isolated through use of restriction endonucleases
Is DNA negatively or positively charged? Why?
Negatively-- phosphate groups
What is the principle behind DNA gel electrophoresis?
using the charge on DNA (-) to move different sized fragments through a gel at different rates--large pieces migrate more slowly
What is the easiest way to denature and re-anneal DNA?
heating and cooling
What is Southern Blotting?
Assay for DNA by: restriction endonuclease digest, then gel electrophoresis, membrane transfer, and visualization with hybridized DNA probes
What is Northern Blotting?
Assay for RNA by: production and isolation of RNA, then gel electrophoresis, membrane transfer, and visualization with hybridized DNA (or RNA) probes
What is FISH?
Fluorescence in situ hybridization. Analysis of DNA (or RNA) expression/presence. Denature DNA in fixed tissue and probe using DNA probes
How is FISH useful to probe for translocation among chromosomes?
You can isolate chromosomes and probe them using labeled DNA probes and see if sections of chromosomes have translocated
What is the Philadelphia Chromosome?
A translocation between chromosomes 9 and 22 that leads to BCR-Ab1 fusion (resulting in chronic myologenous leukemia)
What is RFLP?
Restriction fragment length polymorphism-- differences in digested DNA fragment lengths. These can be electrophoresed for "DNA fingerprinting"
Why is Sir Alec Jeffreys famous?
He was interested in the idea of using repetetive DNA as a source of personal inforamtion (DNA fignerprinting). So there.
Why is Mary-Claire King famous?
she defined genes associated with inherited forms of breast cancer (BRCA1)
How does Sanger Dideoxy (DNA) sequencing work?
using dideoxy nucleotides (ddNTPs) which lack a 3' OH--therefore terminate replication, DNA is synthesized from ssDNA fragments in four reactions (each containing a particular ddNTP at low concentrations); synthesis will stop on select NTP at different lengths-- these can be separated on a gel/capillary and the relative concentrations of nucleotides quantified for sequencing
What is an oligo?
a chemically sythesized oligonucleotide
In what direction are oligos synthesized?
3' --> 5'
What is PCR?
Polymerase Chain Reaction-- DNA replication and amplification technique using DNA primers and Taq polymerase in a temperature-controlled reaction. Used to amplify DNA/analyze expression of DNA
What is a DNA microarray?
Technique using spotted probes and labeled DNA to measure expression levels of RNA in tissues, whatever
How might a microarray be useful in studying cancer?
Determining what genes are upregulated in cancerous tissue--could be used to characterize malignant vs. nonmalignant, test for prognosis of metastasis, analysis of chemo (or other) treatment, determining cancer genes, and more!
How might microarrays be useful in studying develomental anomalies?
difficult-to detect clinically, but may be due to microdeletions/microtranslations, analysis of small changes in DNA may allow more robust diagnostics
What is the goal of pharmacogenetics?
To understand and direct drug targets and drug efficacy based on genetic SNPs in families/individuals
How could pharmacogenetics be used to tailor therapies?
allows genotype-specific drug targeting; eg in cytochrome p450 gene polymorphisms --> disease but gene chip analysis can determine if patients will respond to particular drugs
What is recombination?
the exchange of genetic material between separate DNA duplexes, which may yield distinct products
What genetic processes involve recombination?
meiosis, antibody and T cell diversity, viral integration into genome, transposon integration
What are the two types of recombination?
homologous (genetic) and site-specific
What are the major differences between homologous and site-specific recombination?
DNA length: homologous preserves; site-specific doesn’t; location: homologous occurs between homologous sequences; timing: homologous generally meiosis and repair, site-specific in VDJ, host integration
Is genetic information lost in homologous recombination?
What are the overall steps in homologous recombination?
Initiation: ds break and creation of 2 free ends; pairing "invasion" of homologous DNA; DNA synthesis: polymerization and ligation; resolution of the intermediates
What is NHEJ?
Non-homologous end joining; joining non-homologous ends to repair double-stranded breaks
What does Rec BCD do in recombination?
helicase and endonuclease activity
What does RecA do in recombination?
ssDNA binding protein, mediates invasion during homology search
What is Rad51?
a critical eukaryotic homolog of RecA--loss or failure to assoc. w/ Brca1 or Brca2 can result in accumulation of DNA damage
What do Ruv A, B, and C do in recombination?
promote resolution of the intermediaries (eukaryotic homologues function in apoptosis and chromatin remodeling)
What enzymes are important in homologous recombination?
Rec BCD- ds break and pairing; RecA- ssDNA binding, invasion; Ruv A,B,C- promote resolution
What enzyme is critical for site-specific recombination?
What are the two types of transposition?
transposon (donor DNA-->DNA intermediate-->DNA reintegration) and retrotransposon (Donor DNA-->RNA-->DNA intermediate-->reintegration)
What are the major constituents of a transposon?
flanking (host) DNA, mobile element (w/ regulation/targeting sequences)
What is the difference between replicative and non-replicative (conservative) transposition?
Replicative (involves complex transposons) generates a copy of the transposon and results in 2 copies of the segment through site-specific recombination; non-replicative excises the DNA segment for the transposon and inserts it into a new site in the genome
What is an insertion element
simplest transposon unit--infect prokaryotes and eukaryotes, encode transposase and have inverted terminal repeats, flanked by direct repeats; undergo random integration into genome
What is the difference between an ITR and a DR (in a transposon)?
ITRs are ~50 bps characteristic of IS that mark the boundaries of the IS, DRs are generated because of jagged cleavage of target DNA and insertion, then repair of gaps (they are length, but not sequence-specific)
What are the two functions of transposase in IS translocation?
Makes a ds blunt cut between the ITR and DR of transposon, makes a ds jagged cut in target DNA
What are complex transposons?
additional genes flanked by two ISs, generally infect bacteria, eg provide antibiotic resistance
What are the major differences between insertion sequences and complex transposons?
IS uses non-replicative (conservative) transposition; complex us replicative, IS is moved, complex is replicated through ds nick, ss ligation, replication, and recombination
What is a retrotransposon?
like a complex transposon, but with ability to reverse transcribe its own mRNA to DNA (encode reverse transcriptase) mainly infect eukaryotes; major source of change in eukaryotic genome
What are the two types of eukaryotic nonviral retrotransposons?
What are the differences between LINEs and SINEs
L=Long interspersed elements, S=short; LINEs are transcribed (by RNA PolII)--> have ORFs and generate proteins, SINEs don't, have no ORFs (are transcribed by RNA PolIII)
What are some uses of transposons?
gene disruption, change activity at a promoter, deletions/inversions, insertion of a harmful or advantageous gene
What are Alus and what is L1?
Alus are SINEs (eg NF1) and L1 is a LINE
What are examples of mutagens?
Ionizing or UV radiation, chemical mutagens, pathogens, transposons
What is the difference between ionizing and UV radiation mutations?
UV radiation-->pyrimidine dimers-->ds break vs. x-ray-->???-->ss break
What are the two types of tautomeric shifts in base pairs leading to mutation?
A-->Aimino binds C instead of T; T-->Tenol binds G instead of A
How does 5-Bromouracil lead to bp mutation?
5-BUketo binds to A, but quickly changes to 5-BUenol,, which binds to G
How does bp mutation relate to cacer?
mutations can lead to cancer, but can also be important to block DNA replication/cancer
How does the formation of pyrimidine dimers disrupt DNA?
intra-strant covalent link between thymines (cytosine?) (C6-C or C4-C) distorts the duplex, corrected by excision
How does nitrous acid cause mutations in DNA?
deaminates bases (Adenine-->Hypoxanthine and binds with Cytosine; cytosine-->uracil and binds with adenine)
How can methylation cause mutations in DNA?
methyl group adds onto base (onto O in guanine) and distorts double helix; makes site sensitive to hydrolysis and can cause loss of a base
How does oxidation cause mutations in DNA?
thymine-->thymine glycol (disrupts structure/H-bonding)
What is an example of a bulky adduct that mutates DNA?
cis-platin crosslinks DNA duplexes; adds a platinum product that has two binding sites
What proteins in prokaryotes are involved in DNA repair?
Lex A, Rec A (SOS response)
What proteins are involved in eukaryotic DNA repair?
ATM or p53
How is prokaryotic DNA repair regulated?
Lex A inhibits DNA repair by binding to inhibitory sites; Rec A recognizes DNA damage and cleaves LexA and signals need for repair, Lex A falls off and DNA repair proteins are made
How is eukaryotic DNA repair regulated?
p53 and ATM proteins recognize DNA damage and signal for help, p53 halts the cell cycle and activates DNA repair genes; DNA damage-->ATM-->p53-->DNA repair proteins are activated
What is the difference between DNA damage and mutation?
they are not the same-- DNA can be damaged in a number of ways, but it is in the repair of DNA that the sequence can be permanently altered (many repair mechanisms are imprecise or error-prone)
What are the different repair systems and the enzymes involved?
DNA pol proofreading, DS break repair --NHEJ, mismatch repair, chemical modifications--O6 MGMT, BER; Repair of UV Radiation Damage--NER, Photolyase, Translesion; Post-replication recombination repair
How does DNA Pol I proofreading work?
As DNA Pol synthesizes DNA, proofreads, using 3' --> 5' exonuclease activity; if it recognizes a mismatch, it cleaves it out and replaces it w/ proper dNTP
How does DNA Pol cut out mismatches
Transfers growing strand from polymerization site to exonuclease site, cuts it out, goes back to synthesis
What proteins are involved in NHEJ?
Ku and DNA-dependent kinase bind the free ends of dsDNA and form a synapse-- DNA PKc recruits Rad50/MRE11/NBS1, which chews off the damaged ends
What is the end result of NHEJ?
loss of some base pairs (chewed off until shrot homology regions are found) but religation of DNA
What enzymes are involved in prokaryotic mismatch repair?
Mut S, Mut L, Mut H, exonucleases, DNA Pol III, ligase
What is the sequence of events in prokaryotic mismatch repair?
Mut S dimer binds to the mismatch, Mut L dimer binds to Mut S, sending Mut S translocating to a GATC site, Mut H joins the complex at GATC, cleaving the unmethylated strand. Exonucleases degrade the strand and then DNA Pol III fills in, ligase finishes the job.
What are the eukaryotic enzyme equivalents to prokaryotic mismatch repair?
Mut S = MSH proteins, Mut L = MLH proteins, Exonuclease = exonuclease, DNA Pol III = DNA Pol delta, Ligase = ligase, SSB's = RPA.
What are the major differences between prokaryotic and eukaryotic mismatch repair?
The strand discrimination process is unknown--GATC site isn't used (no Mut H equivalent known)
What are some important mismatch repair defects to know about?
MSH2 defects can have harmful consequences: DNA Pol slippage can create oncogenes, Cis-platin is used to block cancer; MSH2 problems can block this effectiveness.
How is improper methyl modification repaired?
O6 Methyl-Guanine-Methyl transferase transfers the improper methyl group onto a cysteine group on itself
What is Base Excision Repair and how does it work?
A chemically damaged/incorrect base is replaced with the proper one; glycosidase cleaves the base, then AP endonuclease cleaves off the sugar, then DNA Pol fills in the gap, then ligase ligates.
What DNA polymerases are involved in BER?
DNA Pol I (prokaryotes), DNA Pol beta (eukaryotes short patch) DNA Pol delta (eukaryotes long patch)
What is the difference between short patch and long patch BER?
short patch--repair of one improper NT, long patch--repair of multiple improper NTs
What is the sequence of events in eukaryotic DNA repair?
Genotoxic agents/Spontaneous damage/Replication errors-->DNA damage-->cell cycle arrest and repair by BER, recombination, mismatch repair-->mutation (if not repaired) or restored DNA (if repaired) or Apoptosis (if irreparable)
Lecture 12
Finishing Repair
How can DNA damage become a mutation?
if repair mechanisms don't work and the damaged cell does not undergo apoptosis, and the residual damage is carried by a germ-line cell, the damage can become a mutation
What are the main approaches possible to correct DNA damage?
cell cycle arrest, then BER, NER, recombination, mismatch repair, direct reversal, apoptosis as a last resort
What is NER?
nucleotide excision repair; process by which large DNA structural damage is repaired
What proteins are involved in prokaryotic NER?
Uvr proteins
What are the two main types of eukaryotic NER?
global genome repair and transcriptional coupled repair
How much damage can be accomplished by eukaryotic NER?
up to 9 kb of DNA
What is Xeroderma Pigmentosa?
deletion of XP genes-- 8 genes can be affected, causing an inability to repair DNA damage due to UV exposure (more common to get skin cancer)
How does prokaryotic NER work?
Uvr A protein, bound to DNA and scanning, detects deformation of DNA; Uvr A recognizes damage and binds there, Uvr B recruited and binds Uvr A, Uvr A is released in ATP-dependent fashion, Uvr C binds Uvr B at site of damage, Uvr C nicks 3-4 bp on one side, 4 bp on other side, Uvr B and C are released, Uvr D binds damaged area and helicase activity unwinds, releasing damaged strand; DNA Pol I and ligase repair gap
What does Uvr C do?
recognizes Uvr B on damaged DNA and nicks 3-4 bp and 4 bp on either side, then dissociates
What does Uvr D do?
helicase--recognizes damaged and (Uvr C) nicked DNA and unwinds it, freeing damaged strand as oligos that can be degraded
How does eukaryotic global genome repair work?
initial damage recognition, proteins bind and cleave DNA, oligo is removed and DNA re-synthesized to fill gap
What proteins are necessary in eukaryotic global genome repair?
XPC and hHR23B dimer binds to damaged and distorted DNA, TFIIH and other proteins bind to the complex and join site of damage, XPB and XPD are subunits of TFIIH and helicases, RPA and XPA bind and DNA is furtherunwound; XPG and XPF/ERCC1 are nucleases recruited to cleave DNA, DNA pol delta/epsilon fill in gap and ligase seals it
What do XPB and XPD do?
subunits of TFIIH, they are helicases that function in global genome repair
What do XPG and XPF/ERCC1 do?
they are nucleases that celave DNA and remove damaged nucleotides
What DNA pols are important in global genome repair?
DNA pol delta and epsilon
What is transcription coupled repair (TCR)?
a version of eukaryotic nucleotide excision repair; RNA pol can stall when it finds damage in the genome, CSA and CSB displace RNA Pol and recruit TFIIH to damaged DNA
What proteins are different between global genome repair and TCR?
RNA Pol recognizes damage and CSA and CSB bind to replace it, then recruit TFIIH (XPC and hHR23B are damage-recognizing proteins otherwise); from here XPB and XPD are the same (helicases) and XPA and RPA continue to lead down same path.
How is UV damage repaired?
translesion--DNA polymerases repair the damage
What proteins repair UV damage?
DNA polymerases (alternative to normal DNA pols)-- prokayrotes use DNA Pol V (Umu C and Umu D) and DNA Pol IV (Din B), eukaryotes use DNA Pol eta, DNA Pol zeta, Din B homolog
Why is UV damage repair error-prone?
a different subset of DNA pols are used to repair DNA, these have higher rates of misincorporation (but this is still better than having a gap in the DNA)
How does DNA Pol V translesion repair work?
DNA Pol III comes upon a lesion and falls off (cannot read through damage), Umu C and D are recruited to site and form dimer (=DNA Pol V), DNA Pol V is able to polymerize past lesion and replication continues (but Pol often inserts the wrong bases)
How does DNA Pol IV work?
Similar to DNA Pol V translesion repair, but leaves out nucleotides during polymerization, often leading to deletions which may result in frameshifts
What are the three main options for eukaryotic translesion synthesis?
DNA Pol eta, DNA pol zeta, Din B1
What is the process of eukaryotic translesion repair?
DNA Pol stalls at position of DNA damage and falls off, repair DNA pols are recruited
What are the characteristics of DNA Pol eta repair?
usually inserts proper nucleotides
What are the characteristics of DNA pol zeta repair?
requires Rev 1 and is error-prone
What are the characteristics of Din B repair?
insterts (often wrong) nucleotides
What causes xeroderma pigmentosa?
defects in XP genes, inability to repair damage caused by UV rays, higher susceptibility to skin cancer
What causes XP variant (XPV)?
defect in DNA pol eta (RAD30) which incorporates As opposite T-T dimers-- uses error-prone translesion repair. Has same symptoms as XP but excision repair is normal
What is Cockayne Syndrome?
defect in CSA and CSB (of TCR), leads to excessive cell death, neurological degeneration and stunted growth
What is post-replication recombination repair?
repairs ss gaps on a DNA strand whose complementary strand has large adducts, uses second genomic copy of damaged strand to synthesize a complementary one
What are the steps involved in post-replication recombination repair?
DNA Pol III stops at the lesion, Rec A facilitates invasion of the damaged strand into duplex of second chromosomal copy, DNA Pol I fills in the gap with complementary nucleotides
How does post-replication recombination repair work?
a gap is left in a newly synthesized strand where damage has taken place; in a poorly-understood process DNA Pol somehow copies nucleotides from the parental strand to fill in the daughter's gap
define a gene
the DNA containing information needed for some product (may be protein or RNA), also includes control sequences
What is the main enzyme involved in RNA synthesis?
multisubunit enzyme RNA Pol
How is the site of the initiation of transcription numbered?
+1 (downstream is +n, upstream is -n)
What are the two main types of RNA?
coding (mRNA in AA code) and non-coding (never become proteins)
What types of non-coding RNA are there?
rRNAs, snoRNAs (parts of ribosomal complex), tRNAs (translation), snRNAs (splicing), others: miRNAs, siRNAs (diverse functions in X-inactivation, gene expression and chromatin structure)
Does RNA act as a single strand?
No-- there are secondary and tertiary structures of RNA
What are some secondary structures of RNA?
stem-loop (large loop loop with double helical stem region with base pairing), hairpin (larger regions of base pairing with smaller loop)
What are some tertiary structures found in RNA?
pseudoknot, others
What do tertiary structures give to RNA?
specify function, influence stability, regulation (turnover), define phylogeny
Give me an overview of transcription
rNTPs are polymerized in a 5'->3' direction (phosphodiester bond formation catalyzed by RNA Pol), phosphate released in process (rxn direction driven by PPi cleavage)
What is the non-template strand?
the coding strand, since the template is complementary to both the RNA and the non-template
What are the three key steps in RNA transcription initiation?
polymerase binds to promoter sequence, forming closed complex, RNA Pol melts DNA duplex near transcription site, RNA Pol catalyzes formation of phosphodiester bond between two rNTPs
What is the closed complex?
RNA Pol bound to DNA duplex at transcription initiation site-- Pol is not yet working
What is the open complex?
RNA Pol has melted DNA duplex, forming transcription bubble
What is the transcription bubble?
ssDNA made as RNA Pol moves along DNA synthesizing RNA
How does RNA elongation work?
RNA Pol moves down template strand in 3'->5' direction, melting DNA and adding rNTPs to growing RNA chaing-- transient DNA-RNA hybrid exists at region of polymerization
How is transcription terminated?
once a stop site is reached, RNA Pol releases completed RNA and dissociates from DNA duplex
How are prokaryotic genes organized?
operons-- functional clusters of genes, engage in simultaneous transcription and translation
How are eukaryotic genes organized?
generally no clustering (some exceptions e.g. beta-globulin), primary transcript is processed (5' and 3' modifications and splicing) and RNA is transported to different regions of nucleus and cytoplasm
What are the main subunits of prokaryotic RNA Pol?
beta, beta-prime, alpha, omega, (delta-->specificity)
Lecture 13
Eukaryotic Transcription
What does the prokaryotic RNA pol haloenzyme consist of?
beta, beta prime, 2 alphas, omega (delta)
What is the core enzyme?
the beta and alpha subunits
What is the specificity/initiation subunit?
What subunit is necessary for loading?
Is eukaryotic RNA pol more or less complicated?
Do yeast and bacterial RNA pols have similar stuructures?
yes-- but yeast has 10 additional subunits that are not part of bacterial pol
Are eukaryotic genes organized into operons?
What is an operon?
a group of genes contolled by one common promoter (operator)
What in eukaryotic transcription replaces the function of the delta subunit in prokaryotic transcription?
multiple transcription factors (TFs) and TBP (TATA binding protein)
What are some examples of promoters?
GC-rich region, box A, box B, box C, TATA box

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