Intro Biochem 527 Exam 4

Transcription, RNA Metabolism, RNA Processing

transcription

DNA-dependent synthesis of RNA

130

the human genome contains genes for >________ repair proteins

lesion

DNA damage

mutations

a random error in gene replication that leads to a change

\- if unrepaired, lesions become this

\
ex) substitutions (point mutations), deletions, additions

silent mutations

this mutation has around no effect on gene function or affects a nonessential region of the DNA

types of DNA damage

mismatches, abnormal bases, pyrimidine dimers, backbone lesions

mismatches

type of DNA damage that arise from occasional incorporation of incorrect nucleotides

abnormal bases

type of DNA damage that arise from spontaneous deamination, chemical alkylation, or exposure to free radicals

pyrimidine dimers

type of DNA damage that forms when DNA is exposed to UV light

backbone lesions

type of DNA damage that occurs from exposure to ionizing radiation and free radicals

methylation of DNA and repair

\- Newly synthesized strand is unmethylated for a short period after synthesis

\- any replication errors must reside in the unmethylated strand

\- Methyl-directed mismatch repair system will cleave the unmethylated strand in the initial part of the repair process

cancer

Accumulation of mutations in eukaryotic cells are strongly correlated with ________

Mismatch repair

The cellular process that uses specific enzymes to remove and replace incorrectly paired nucleotides.

\
1) MutL and MutS proteins recognize methylated GATC

2) MutH binds to MutL-MutS-DNA complex, making a DNA loop

3) DNA strands thread through the complex until methylated GATC is encountered

4) MutH cleaves the nonmethylated DNA strand on the 5’-side of the G

5) DNA unwinds and is degraded 3’ → 5’

6) Removed sequence is replaced using DNA Pol III and DNA ligase

7) Cleaved strand with incorrect nucleotide is degreaded by exonucleases

8) Cleaved strand is then rebuilt by DNA Polymerases and sealed with DNA ligase

mutagens

A chemical or physical agent that interacts with DNA and causes a mutation.

* most carcinogens are also this

MutL

"linker protein" in mismatch repair, recruits MutH to MutS

MutS

In mismatch repair, Recognizes single base mismatches

MutH

In mismatch repair, cleaves the unmethylated strand on the 5'-side of the G in the GATC sequence

GATC

What sequences are methylated in mismatch repair?

3' -> 5'

Which direction is DNA unwound and degraded in mismatch repair: 5' -> 3' or 3' -> 5'?

toward

Helicase II (UvrD helicase), SSB, and exonucleases work to degrade the nonmethylated DNA in mismatch repair which way: towards or away from the mismatch?

base excision repair

DNA repair that first excises modified bases and then replaces the entire nucleotide

* uses specific glycosylases

\
1) DNA glycosylase recognizes specific lesions

2) N-glycosyl bond is cleaved between sugar and base, creating an apurinic/apyrimidinic (AP) site

3) Uracil glycosylate removes uracil from DNA

4_ Other glycosylates make AP sites at 8-hydroxyyG, hypoxanthine, 3-methyladenine, and so on

N-glycosyl

Which bind is cleaved between the sugar and the base in base excision repair by DNA glycosylases?

AP sites

Apyrimidinic or apurinic sites containing no base that can occur spontaneously in DNA

* formed by the cleavage of the N-glycosyl bond between the sugar and base

Uracil glycosylase

glycosylase that removes uracil from DNA

* important bc C spontaneously deaminates to U

C

Which base spontaneously deaminates into U within DNA, and is usually fixed by base excision repair: A, T, G, or C?

repair at AP sites in bacteria

this repair includes:

1) Entire nucleotide is ultimately removed, not just the damaged base

2) AP endonucleases cut the DA backbone around the AP site and removes DNA

3) DNA Pol I synthesizes new DNA

4) DNA ligase seals the nick

AP endonucleases

In base excision repair, this endonuclease cuts the DNA backbone around the AP site, remove DNA

nucleotide excision

DNA damage that distorts the double helix, including abnormal bases, modified bases, and pyrimidine dimers

* pathway involves removal of DNA segment by exinucleases

\
Lesions include:

* pyrimidine dimers (formed by UV light)
* 6,4-photoproducts (from UV light)
* benzo\[a\]pyreneguanine (from cigarette smoke)

exinucleases

in nucleotide excision repair, cuts 2 phosphodiester bonds in a single polynucleotide chain

ABC exinuclease

excision of a lesion-containing oligonucleotide in nucleotide excision repair

* contains UvrA, UvrB, and UvrC
* hydrolyzes the 5th bind on the 3’-side of the lesion and 8th bond on the 5’side
* removes 12-13 nucleotides

Nucleotide excision repair in bacteria

this excision repair includes:

1) Exinculeases cleave DNA backbone in 2 places

2) ABD exinucleases contain UvrA, UvrB, and UvrC that removes 12-13 nt

3) DNA Pol I and DNA ligase to replace the DNA and seal the gap

Nucleotide excision repair in eukaryotes

this excision repair includes:

1) Hydrolyze the 6th bond on the 3’-side and 22nd bond on the 5’-side of the lesion

2) Removes 27-29 nt

3) In humans, gap filled using DNA Pol e and DNA ligase

12-13

How many nucleotides are removed in bacterial nucleotide excision repair by the ABC exinuclease?

27-29

How many nucleotides are removed in eukaryotic nucleotide excision repair?

DNA Pol e

Which DNA polymerase is responsible for filling the gap in nucleotide excision repair in humans after nucleotides are removed?

direct repair

DNA repair in which modified bases are changed back into their original structures

\
ex) ex) Photolyases, O6-methylguanine-DNA methyltransferase, and AlkB

photolyases

use visible light energy to separate UV-induced pyrimidine dimers (repairs pyrimidine dimers)

\- NOT found in humans and placental mammals

O6-methylguanine-DNA methyltransferase

In direct repair, repairs methylated guanine

AlkB

In direct repair, Directly repairs Alkylated Bases:

demethylates 1-methyladenine and 3-methylcytosine

direct repair with photolyase

this type of direct repair includes:

1) Blue light photon (300 to 500 nm) is absorbed by MTHFpolyGlu

2) The excitation energy passes to FADH- in the active site

3) The excited flavin (+FADH-) donates an electron to the pyrimidine dimer to generate an unstable dimer radical

4) Electron rearrangement restores monomeric pyrimidines

5) Electron is transferred back to the flavin radical to regenerate FADH-

T

T/F: O6-Methylguanine leads to mutations by methylating the oxygen in Guanine, thus disrupting the hydrogen bonding between guanine and Cytosine

T

T/F: an unrepaired lesion can cause the replication fork to stall

repair with no undamaged DNA as template

Repair using:

* another chromosome as template (recombination)
* nonhomologous end joining (NHEJ)
* error-prone translesion synthesis (TLS)

translesion synthesis

A damage-tolerance mechanism in eukaryotes that uses bypass polymerases to replicate DNA past a site of damage

* allows DNA to be synthesized with no template

error-prone TLS in bacteria

\- Part of the "SOS" response

\
1) SOS proteins inluced UvrA, UvrB+, UmuC, and UmuD

2) Cleaved UmuD’ and UmuC bind with RecA to create DNA Pol V

SOS response

response system when DNA damage is extensive

SOS proteins

These proteins include:

* UvrA
* UvrB
* UmuC
* UmuD

unmutable

aka. Umu__

\
Lack of these genes abolishes the error-prone TLS pathway

RecA

This protein must be active in order for SOS induction to happen; Cleaved UmuD' and UmuC binds to RecA to create DNA Pol V

V

Which DNA polymerase can process past the damaged area in error-prone TLS: I, III, or V?

TLS polymerases

Most of these polymerases recognize specific types of damage and have appropriate responses, and are limited to short regions of DNA (minimizing mutagenic potential)

\
ex) DNA Pol n (eta) assists when T-T dimer present

short

Most TLS polymerases are limited to ________ regions of DNA, minimizing mutagenic potential: Long or short?

DNA recombination

rearrangement of DNA sequences by exchanging segments from different molecules

* within a chromosome
* from one chromosome to another

\
Involved in many biological processes:

* repair of DNA
* segregation of chromosomes during meiosis
* enhancement of genetic diversity

\
In sexually reproducing organisms, one of the 2 driving forces of evolution

\
This process of co-infecting viral genomes may enhance virulence and provide resistance to antivirals

recombination and mutations

What are the 2 driving forces of evolution in sexually reproducing organisms?

rearrange

In DNA recombination, segments of DNA can _______ their locations within a chromosome, or from one chromosome to another

3 classes of recombination

classes of this process include:

1) Homologous/general recombination

2) Nonhomologous end joining

3) site-specific recombination

homologous/general recombination

Exchange between 2 DNAs that share an extended region of similar sequence

\
Has 3 main functions:

1) assists in DNA repair

2) Links sister chromosomes to properly segregate them between self and daughter cells (Eukaryotic cells)

3) Source of DNA exchange and therefore genetic diversity

homologous

What kind of recombination is responsible for genetic diversity: homologous or nonhomologous?

bacterial homologous recombination

This recombination includes:

1) Replication fork encounters damage in template strand

2) Replication fork collapses due to creation of a double-strand break (DSB)

3) 5’-ending strand is degraded

4) 3’-single-stranded extension is bound by a recombinase, pairs with complementary sequence in intact duplex DNA, and “invades” duplex

5) Creation of branched structure of 3 strands

6) Branch moves to create X-like structure known as “Holliday junction” or “Holliday intermediate”

7) Nuclease and ligase restore the structure of the replication fork and processes the DNA ends

recombinase

an enzyme that promotes genetic recombination

\- binds to the 3'-single-stranded extension in bacterial recombination

Holliday junction

cross-shaped structure that forms during the process of genetic recombination, when two double-stranded DNA molecules become separated into four strands in order to exchange segments of genetic information.

5’

Which ending of a strand of DNA is degraded in bacterial recombination: 3' or 5'?

RecBCD enzyme

Nuclease/helicase that directly loads the RecA protein onto single-stranded DNA overhangs.

\- processes the DNA ends in bacterial recombination

\- binds to broken end and then unwinds and hydrolyzes DNA

1\) RecB moves along one strand 3' -> 5'

2\) RecD moves along other strand 5' -> 3'

3\) RecC subunit binds to the chi sequence (GCTGGTGG) and alters the activity of the enzyme complex

4\) The chi sequence on the 3'-ending strand binds tightly to RecC

5\) The 5'-ending strand continues to be degraded

6\) RecA recombinase becomes active via the assembly of thousands of subunits

7\) Subunits assemble cooperatively on the DNA in steps of nucleation and extension

RecC subunit in the RecBCD enzyme in bacterial recombination binds to the ________ sequence (GCTGGTGG) and alters the activity of the enzyme complex

chi

The RecBCD enzyme acts as a ________/________ that processes the DNA ends at the end of bacterial recombination

nuclease/helicase

chi sequence

short stretch of DNA in the genome of a bacterium near which homologous recombination is more likely than expected to occur.

\- >1,000 of this sequence in E. Coli

\- binding of RecC to this sequence halts 3'-enidng strand's movement through complex of RecBCD enzyme

RuvAB

This complex promotes branch migration via:

\- RuvA binds where four arms of Holliday junction come together

\- Hexamers of RuvB bind opposite arms and use ATP to propel DNA outward

Which part of the RuvAB complex binds to where the four arms of the Holliday junction come together: A or B?

A

RuvC

Nuclease that binds to RuvAB complex, and cleaves Holliday intermediate on opposite sides

Whic Ruv is responsible for cleaving the Holliday junction/intermediate on opposite sides: A, B, or C?

C

nonhomologous end joining

A quick-and-dirty mechanism for repairing double-strand breaks in DNA that involves:

\- broken chromosome ends are simply processed and ligated back together

\- no conservation of DNA sequence

\- this form of repair typically occurs when the chromosomes are condensed and constrained, thus the ends are near each other

1\) Ku70 ad Ku80 proteins bind the DNA ends followed by other proteins - DNA-PKcs (kinase) and Artemis (nuclease)

2\) Artemis removes 5' or 3' single-stranded nucelotides or hairpins that have formed

3\) Helicase separates the ends

4\) Remaining nick is sealed by complex consisting of XRCC4, XLF, and DNA ligase IV

Ku70 and Ku80 proteins

Proteins that bind to the ends of DNA in nonhomologous end joining

DNA-PKcs

Kinase that in complex with Ku proteins, recruits and phosphorylates Artemis (nuclease) in nonhomologous end joining

Autophosphorylates in several locations, including Artemis

Artemis nuclease

Nuclease that is phosphorylated by DNA-PKcs (kinase) in nonhomologous end joining

\- removes 5' or 3' single-stranded nucleotides or hairpins that have formed

Nonhomologous end joining repair typically occurs when the chromosomes are either: condensed or loosened?

condensed

T/F: Nonhomologous end joining is an ideal form of cellular repair

F