BI213: Exam II

during replication

When does DNA repair fix mistakes (chemical changes)?

throughout life

spontaneous, radiation, chemicals

When does damage occur? How does it occur?

mutations in codons, blocking replication/transcription, recombination, DNA rearrangement

What are some potential consequences of DNA mistakes?

Deamination

cytosine becomes Uracil (only found in RNA, so it should not be in DNA)

What is DNA damage that results in the loss of am amino group? What change does this result in?

covalent linkage between two adjacent thymines occurs (forms a thymine dimer)

almost all types of skin cancers

What DNA damage results from exposure to UV light?

What kinds of cancer does this often cause?

bulky group is added to Guanine

80-90% of lung cancers

What DNA damage results from a reaction with a carcinogen?

What does this cause?

Direct reversal of DNA damage - most limited because have to recognize specific kind of damage

photoreactivation of thymine dimers; prokaryotes only (bacteria and some plants - NOT HUMANS)

What the most uncommon type of DNA repair? Why?

What is an example? What type of cells does this occur in?

light and photoreactivating enzyme breaks the cyclobutane ring, repairing DNA

Describe the process of direct repair of thymine dimers?

1. Base-excision repair: repair of Uracil

2. Nucleotide-excision repair: repair of most damage

3. Mismatch repair: replication errors

What are the three sub-categories of excision repairs? What do they each correct?

- DNA containing uracil formed by the deamination of C

- DNA glycosylase recognizes and removes the BASE at the Apyrimidinic (AP) site

- AP endonuclease cleaves DNA at location that lacks a base

- Deoxyribosephosphodiesterase removes the deoxyribose after it has been cleaves

- DNA polymerase and Ligase synthesizes strand

Describe the process of base-excision repair.

both

Does base-excision repair and nucleotide excision repair occur in prokaryotes or eukaryotes

1. damage recognition and nuclease cleavage by Excinucleoase UvrA,B,C in E. coli (complex of 3 proteins)

2. hydrogen bonds removed by helicase

3. bases removed to left and right of damage (12-13 bases) - oligonucleotide is excised and degraded

4. DNA polymerase I (E. coli only) and ligase put in new nucleotides

Describe the process of nucleotide-excision repair of thymine dimers in prokaryotes

Xerodermina pigmentosum XP (skin cancer) - hereditary condition where repair of damage can't occur

How were the enzymes involved in nucleotide excision repair discovered?

1. damage recognition by XP proteins

2. Helicase activity

3. many proteins involved, endonuclease activity results in cleavage and excision of oligonucleotide

4. DNA polymerase δ and Ligase involved in DNA synthesis

Describe nucleotide-excision in mammalian cells

7 different genes associated with xeroderma pigmentosum (XP)

What does nucleotide-excision repair involve in mammalian cells?

10^-2

What does selection by complementary base pairing reduce error rate of DNA replication to?

10^-5

What does base selection by DNA polymerase reduce the error rate of DNA replication to?

10^-7 to 10^-8

What does proofreading by DNA polymerase reduce the error rate of DNA replication to?

less than 10^-9

What does mismatch repair reduce the error rate of DNA replication to?

how to recognize the newly synthesized strand

What is the trick for improving the fidelity of DNA replication?

During DNA replication

When does mismatch repair occur?

1. Old strand has methyl groups scattered throughout

2. Mismatched base is recognized by MutS

3. MutH cleaves opposite of a methyl group

4. MutS, MutL, helicase and exonuclease together directly excise between the nick and the mismatch

5. DNA polymerase and ligase fill the gaps

Describe the process of mismatch repair in E. Coli.

MutS and MutL, but do not have MutH

What mismatch repair enzymes do eukaryotes have? Which ones do they not have?

Mutations in MutS and MutL

What is responsible for hereditary colorectal cancer affecting 1 in 200 people (about 15%)?

- the presence of single strand breaks in newly replicated DNA - the ends of growing strands (not methylation)

What dictates strand specificity in mismatch repair in eukaryotic cells?

Hereditary nonpolyposis colorectal cancer (HNPCC)

What does mutations in mismatch repair cause?

1. Break is recognized on leading strand or in gap between okazaki fragments (why we don't need the MutH protein- pre-cleaved) by nicks

2. MLH and MSH bind, excision between mismatch and nick

3. replication continues by (DNA polymerase ε in leading and DNA polymerase δ in lagging strand)

Describe the process of mismatch repair in mammalian cells

at the replication fork

Where does translesion repair occur?

DNA polymerase I

Polymerase III

Polymerase II, IV and V

In E. Coli:

What DNA polymerase is responsible for filling gaps/repair?

What DNA polymerase is responsible for DNA replication?

What DNA polymerase is responsible for translesion repair?

Polymerase a + primase

Polymerase d

Polymerase e

Polymerase b & at least 4 others

In eukaryotes:

What DNA polymerase is responsible for primer synthesis?

What DNA polymerase is responsible for replication, filling gaps, and repair?

what DNA polymerase is responsible for replication?

What DNA polymerase is responsible for translesion repair?

- problem at replication fork, no longer have undamaged opposite strand for excision repair

- normal DNA polymerases recognize some forms of damage, not normal bases

Where and why is translesion DNA synthesis necessary?

error prone, but can insert correct bases opposite a mutation/lesion

What is one problem with translesion DNA repair?

both prokaryotes and eukaryotes

What does translesion repair occur in?

1. Normal replication blocked by thymine dimer

2. specialized DNA polymerase targeted to lesion (highly related)

3. DNA synthesis across lesion by specialized (less accurate) DNA polymerase

4. Resumption of normal replication by regular DNA polymerase

5. Old strand still has damage - so use excision repair after tranlesional

Describe the process of tranlesion DNA synthesis.

Double-strand breaks; during or after DNA replication

What is severe damage that results in a break of both strands? When does it occur?

non-homologous end joining:

1. Ionizing radiation causes a double-strand break

2. nonhomologous end joining - immediate ligation by enzymes

3. bases lost

What kind of repair fixes double stranded breaks during replication?

Describe the steps.

recombinational repair of double strand breaks

What DNA repair pathway is responsible for hereditary breast cancer?

homologous recombination - they remain associated

What type of double strand repair occurs after replication? What happens to the daughter cells?

- BRACA2 (breast cancer gene) recruits RecA/Rad51 which binds to ssDNA at break

- nuclease digestion and strans invasion occurs (sequence exchange)

- DNA synthesis and ligation

Describe the process of homologous recombination

immunoglobulin

10^11 different antibodies

What can produce antibodies against any foreign protein?

How many different antibodies can be produced?

two types of polypeptides (2 light chains and 2 heavy chains) connected by disulfide bonds

made up of a constant region and variable region on both chains

Describe the structure of an immunoglobulin?

3

2

1

How many different genes produces antibodies?

How many genes produce light chains?

How many genes produce heavy chains?

site-Specific recombinations

How is immunoglobulin diversity produced?

- numerous V and J regions (somewhat like (open reading frames) and a constant regions

What are the components of the germ-line DNA that produce immunoglobulin light chains?

- in the B lymphocyte, a unique combination of V and J regions along with the constant region is made (the rest are excised and destroyed)

- gene is transcribed and then spliced, producing immunoglobulin mRNA

Describe site-specific recombination

no, it will produce the same antibody for its lifetime, although it made be dormant for some amount of time

Once a B-Cell begins producing an antibody, can it switch?

-constant region

- V, J, and D regions

they have the additional D region - V(D)J region

What is the structure of the genes that code for immunoglobulin heavy-chain genes?

Why are they larger than light chains?

RAG1 and RAG2

cleavage and joining at recombination sites (join to recognize RS sites and catalyze recombination)

What does the V(D)J recombination mechanism involve for both light and heavy chains?

What is the function of these enzymes?

DNA (transcription) - RNA (Translation) - Protein

Regulation of gene expression

What is the centra dogma of biology?

What is critical for this to be successful?

transcription

What is the primary form of regulation gene expression?

3' to 5'

5' to 3'

In what direction does RNA polymerase read in transcription?

In what direction are nucleotides added on the mRNA?

"from the beginning or "from scratch" - meaning it doesn't need a primer

What does it mean that RNA polymerase can initiate de novo?

s (sigma)

What finds the right DNA sequence (promotor) for the initiation of transcription in E.Coli?

5 subunits required for elongation

- s subunit required for promotor recognition

What does core RNA polymerase consist of in E.Coli?

promotor

What is the site to which RNA polymerase binds to initiate transcription?

+1

-35 and -10 (the TATA box)

What is the number of the transcription start site on E.Coli promotors?

What are the binding sequences for sigma on E.Coli promotors?

1. Initiation - sigma searches for promotor and binds

- closed promotor becomes open at +1 causing unwinding of about 12 bases

2. Elongation (add ribonucleotides) - after 10 bases sigma is released, which indicates the elongation phase

3. Termination signal

What are the steps of transcription?

joining of 2 NTPs

How does sigma bind?

stem-loop in RNA (formed by mirror images) causes dissociation from DNA

What is the most common form of transcription termination?

inverted repeats

What forms mirror images that lead to stem-loops?

3, one RNA polymerase could not handle all the different transcriptions that need to be made

How many RNA polymerases are there in eukaryotes?

Why are there more than one?

have the same enzymatic activity (synthesize RNA from DNA) but they have target gene specificity

What is the same about RNA polymerases I, II, and III? What is different?

- they have different combinations of core promotor elements (6)

How do different genes differ in the formation of polymerase II preinitiation complexes in eukaryotes?

TFIID and TFIIB

What transcription factor(s) recognizes promotor elements in eukaryotes?

general transcription factors (TFIIX)

What bind to core promotor elements and are required for basic transcription (multi-subunit protein complexes)

TBP

What is the TATA box binding protein?

TFIID, TFIIB, TFIIF, RNA polymerase II, TFIIE, TFIIH

What is the order of binding in the polymerase II preinitiation complex in vitro?

5

How many general transcription factors are minimally required for initiation in vitro?

regulating activity

7

52 repeats in humans

serine

What is CTD responsible for?

How many many positions does it have?

How many repeats are in humans?

What is in the 5th spot?

CTD kinase

What does TFII H include?

carboxyl terminal region (not RNA)

What is CTD?

mediator stimulates basal transcription and facilitates interaction of polymerase complex with gene specific TFs

What occurs in eukaryotic transcription after the TFIIX factors form the complex and RNA polymerase II binds?

gene-specific TFs

What is not needed in a test tube but is necessary in the nucleus to determine rate of transcription?

TFIIH - protein kinase (serine-5 phosphorylated)

What releases polymerase in transcription?

What is it and how is it phoshporylated?

protein that phosphorylates target protein

What is a kinase?

CTD

What is phosphorylated to initiate transcription?

downstream of their promotors

Where are a large number of genes stalled until appropriate signals are received?

every 50 nucleotides

About how often is transcription paused at a checkpoint?

negative regulators

What are proteins that bind to RNA Polymerase II that pause transcription?

PTEF-b Kinase

restarts RNA polymerase II

What phosphorylates negative regulators to inhibit them?

What does this do?

RNA Polymerase I

What is the primary RNA polymerase responsible for ribosomal RNA

18S, 5.8S, 28S

What are the Ribosomal RNA genes (rDNA)

three

one gene/ONE promotor

How many distinct rRNAs are there? How many genes/promotors?

tRNA and snRNA

What are other examples of RNA genes?

RNA polymerase III

What regulates and transcribes other non-coding RNAs (snRNAs and tRNAs)

gene responsible for making an enzyme to digest lactose

promoter (P) + operator (o) + structural genes (z, y, a)

What is the lac operon?

What are the components of an operon?

cis-acting control sequences

What makes up the operator on the lac operon?

when lactose is absent- a repressor binds to the operator (specific DNA sequences) and transcription is blocked

when lactose is present, it binds to the repressor deactivating it so transcription proceeds

regulation is by lactose levels

Describe the negative control of the lac operon?

glucose (lactose is the alternate nutrient)

In addition to lactose levels, what is the lac operon also regulated by?

- When glucose is high, the positive activator is inhibited (cAMP is not produced, so Catabolite Activator Protein (CAP), the activator) is not turned on)

- if glucose is low, cAMP is produced from ATP and CAP is active, so the lac operon is turned on

Describe the positive control of the lac operon.

regulatory sequences, positive and negative

What are extremely important for transcription in eukaryotes?

- discovery of a new gene, want to identify the promotor:

1. Introduce DNA into cultured animal cells (select for stably transformed/molecular clone)

2. look for the TATA box to try and locate the promotor

3. With the suspected regulatory sequence, add a special reporter gene so you can detect it

4. Transfect recipient cells; lyse and test for expression of the reporter gene

Describe the luciferase reporter assay experimental method for the identification of eukaryotic regulatory sequences

firefly luciferase - makes glow

What is an example reporter gene?

- amount of luciferase units is correlated to the amount of transcription that occurs (non will happen without the promotor)

- the control is just the plasmid without suggested regulatory sequence

What do the results of a luciferase reporter assay suggest?

a eukaryotic promotor

DNA synthesis

What is the promotor of the herpes virus thymidine kinase gene?

What is it responsible for?

-25

+1

At what position of the eukaryotic promotor is the TATA box?

At what position is the mRNA transcription initiated?

other cis-acting sequences (binding for gene specific transcription factors)

What, in addition to a promotor, is required for efficient transcription?

enhancers

help control transcription of many mammalian genes

What are cis-acting regulatory sequences that are farther away from transcription start sites?

What do they do?

- upstream from the gene (5' end)

- Downstream (3' end) from start site

- flipped in opposite direction and still enhancing

Where do enhancers bind gene specific transcription factors?

more than 50 kb (there can even be other genes involved)

How far from promotors can enhancers be in mammalian cells?