Chapter 9 MCB

Can be recognized and cleaved by a specific AP endonuclease

Base Excision Repair: Can be recognized and cleaved by a specific __ __________

Specialized enzymes can recognize specific damaged bases and remove them without breaking the phosphodiester bonds in the DNA backbone

Base Excision Repair: Specialized enzymes can recognize _____ ______ ______ and remove them _______breaking the phosphodiester bonds in the DNA backbone

The result is an abasic site, or AP site (apurinic or apyrimidinic).

Base Excision Repair: The result is an ______ ______, or _ _ site (apurinic or apyrimidinic).

This AP site allows DNA Pol I to synthesize a replacement strand containing the proper base

Base Excision Repair: This AP site allows _____ _______ ____ to synthesize a replacement _______containing the proper _______

consist of repeats and spacers that do not encode proteins, but near them lie CRISPR-associated gene families that do encode proteins

CRISPR: consist of _____and _____ that do not encode_____, but near them lie _______-______ _____ _____ that do encode proteins

immune system

CRISPR: is a primitive microbial____ system

F+ receptors F-

Conjugation: 1. sex pilus from the __ plasmid donor attaches to ______ on the recepient cell or ___

relaxosome bridge

Conjugation: 2. contraction of the pilus draws the 2 cells together and forms a ____ ______

relaxase nicks DNA at oriT (nic site)

Conjugation: 2a. relaxase ____ DNA at _____

5' end begins transfer through the bridge

Conjugation: 3. The F factor is nicked at oriT, and the __ end begins transfer through the ______

the strand remaining in the donor is replicated by DNA polymerase

Conjugation: 4. the strand remaining in the donor is ______ by ____ ______

5. once in the recipien, the transferred strand circulizes and is replicated using RNA primers and DNA polymerase since it is the lagging strand

Conjugation: 5. once in the recipient, the transferred strand ______ and is ______ by using ____ primers and DNA polymerase since it is the lagging strand

6. the recipient has been converted to a donor

Conjugation: 6. the _____ has been converted to a _____

fertility factor ( F factor)

Conjugation: A well-studied example of a transferable plasmid in Escherichia coli is the

TraJ, TraH, TraI (relaxase), TraK, TraG

Conjugation: Relaxosome is made up of:

begins with contact between the donor cell, called the F+ cell, and a recipient F- cell.

Conjugation: begins with contact between the donor cell, called the___cell, and a recipient____ cell

transferable plasmids

Conjugation: contain all the genes needed for pilus formation and DNA export

oriV oriT

Conjugation: fertility factor (F factor) has 2 replication origins, what are they?

transferable plasmids

Conjugation: requires the presence of special ______ ______

oriT

Conjugation: used during DNA transfer

oriV

Conjugation: used in nonconjugating cells

Inversion

DNA is flipped in orientation

Reversion

DNA mutates back to original sequence

1. Error-proof repair pathways (prevent mutations) 2. Error-prone repair pathways (which risk introducing mutations)

DNA repair is divided into two types:

nucleotide residues interfere with polymerase funtion and stop replication or transcription

Damage caused by ROS: 1. ROS can modify ____ _____ 2. modifications can interfere with the ____ ____ and stop replication or _____

Base modifiers

Ex. Nitrates, nitrites

1. Point Mutation 2. Insertion and Deletion 3. Inversion 4. Reversion

Forms of Mutation: 4

1. spacer acquisition 2. crRNA processing 3. effector stage

Function of CRISPR in 3 stages:

RuvC

Generalized Recombination: Ligation and cleavage

1. closed circular molecule + linear molecule 2. single linear molecule= bacterial death ( can be saved by 2nd crossover) 3. single large circular molecule= cointegrate molecule

Generalized Recombination: Ligation and cleavage (by RuvC) can result in three endpoints

RecA filament

Generalized Recombination: RecA binds to free nicked end =

RecA molecules are able to scan DNA molecules for homology and align the homologous regions, forming a triplex DNA molecule, or synapse

Generalized Recombination: RecA molecules are able to ___ DNA molecules for ____ and align the homologous regions, forming a _____ ____ ____, or _______

Deinococcus radiodurans is thought to use RecA protein to reconstruct the chromosome after extreme radiation damage

Generalized Recombination: _______ ______ is thought to use RecA protein to reconstruct the chromosome after extreme _____ damage

protein called RecA

Generalized Recombination: central player in generalized recombination is a ____ called _____

RecA

Generalized Recombination: finds homology and mediates strand invasion (crossover)

RuvAB

Generalized Recombination: mediates branch migration (extending crossover)

RecBCD

Generalized Recombination: unwinds donor dsDNA to chi site, nicks strand, then continues to unwind

Recombination

Genetically-engineered DNA molecule formed by splicing fragments of DNA from a different source or from another part of the same source, and then introduced into the recipient (host) cell.

1. base analogs 2. base modifiers 3. intercalators

How do chemical agents cause mutations ? 3

n the Ames test, we're actually testing whether a chemical can mutate the mutation back to normal.

In the Ames test, we're actually testing whether a chemical can mutate the mutation back to normal.

-use indiscriminate DNA as food -use specific DNA to repair damaged genomes -acquire new genes through horizontal gene transfer

Know why bacteria would undergo natural transformation (3)

specific protein complexes called transformases

Many cells are capable of natural transformation: --require what?

MutS

Methyl Mismatch repair: 1. _____ binds DNA mismatch

MutHL

Methyl Mismatch repair: 2. MutS draws _____ near the GATC site

MutL

Methyl Mismatch repair: 3. ____ recognizes the methylated strand (on parental strand) and bring it in a _____

MutH

Methyl Mismatch repair: 4. _____ cleaves the unmethylated strand 5' to the GATC

UvrD exonucleases DNA polymerase 1

Methyl Mismatch repair: 5. ______ unwinds the nicked strand, ______ nucleases remove the damaged strand, and _____ ______ _ synthesizes a replacement

1. Silent Mutation 2. Missense Mutation 3. Nonsense Mutation 4. Frame-shift mutation

Mutations can be categorized into information classes: (4)

mutagens

Mutations can be caused by _______

UvrA and UvrB

Nucleotide Excision Repair steps: 1. _____ and ____ form a complex

UvrAB damaged

Nucleotide Excision Repair steps: 2. the _____ complex binds to ______ DNA

UvrA

Nucleotide Excision Repair steps: 3. _____ bends the DNA

UvrA

Nucleotide Excision Repair steps: 4. ____ is ejected

UvrB UvrC

Nucleotide Excision Repair steps: 5. _____ lures the actual cleaving enzyme, _____ to the site

UvrC 2

Nucleotide Excision Repair steps: 6. _____ cleaves the phosphodiester backbone of the damged strand at ___ places

UvrD mononucleotides

Nucleotide Excision Repair steps: 7. _____ helicase strips the single-stranded fragment containing the mutation from the DNA Damaged DNA is degraded to _________

DNA Pol 1 DNA Ligase

Nucleotide Excision Repair steps: 8. _____ _____ __ fills the gap and ___ ___ seals the new DNA to the 5' end of the preexisting strand

Error-prone repair pathways

Operate only when damage is so severe that the cell has no other choice but to die

-perturbing the membrane by chemical (CaCl2) or electrical methods (electroporation)

Others require artificial manipulations: -perturbing the membrane by chemical ______ or _________methods (__________)

The enzyme photolyase binds to the pyrimidine dimer and cleaves the cyclobutane ring

Photoreactivation The enzyme ______ binds to the pyrimidine dimer and cleaves the _______ _____

An organism that manages to survive a phage attack captures a piece of the invader's genome and wields it as defense against future attack

Purpose of CRISPR: An organism that manages to survive a ______ attack _____a piece of the invader's ______and wields it as ______ against _______ attack

thymine dimer

Recombinational repair: 1. replication fork approaches ______ ______

DNA pol

Recombinational repair: 2. ____ _____ skips damaged region

RecA double

Recombinational repair: 3. the ____ protein binds to the sister ____ helices at the single stranded segment

RecA-dependent recombination

Recombinational repair: 4. _____-______ _______ replaces the damged strand gap with a section of undamaged strand

Gap DNA pol

Recombinational repair: 5. ___ in undamaged strand is repaired by ____ _____

pyrimidine dimer

Recombinational repair: 6. the ____ ____ can now be repaired by normal NER or other repair pathways

A single-stranded segment of the undamaged daughter strand can be used to replace a gap in the damaged daughter strand. carried out by RecA

Recombinational repair: A single-stranded segment of the ______ ________ strand can be used to replace a gap in the damaged daughter strand. carried out by ______

It will work on any damage that causes gaps during replication

Recombinational repair: It will work on _____ damage that causes ____ during replication

Takes place at the replication fork

Recombinational repair: Takes place at the ______ ________

is not limited to pyrimidine dimers

Recombinational repair: is not limited to _______ ________

Photoreactivation

Removes pyrimidine dimers from UV irradiation

does not utilize RecA and moves only a limited number of genes

Site Specific Recombination: does not utilize ____ and moves only a ____ number of genes

involves very short regions of homology between donor and target DNA molecules

Site Specific Recombination: involves very ____ regions of homology between donor and target DNA molecules

recognized specifically by dedicated enzyme systems, which catalyze a crossover between them to produce a cointegrate molecule

Site Specific Recombination: this homology is recognized specifically by dedicated _____ systems, which catalyze a _____between them to produce a _____ molecule

Spontaneous mutations are rare because of the efficiency of DNA proofreading and repair

Spontaneous mutations are _____because of the efficiency of DNA _____ and _____

effector stage

Stages of CRISPR: CRISPR RNAs guide bacterial molecular machinery to destroy the viral material. Because CRISPR RNA sequences are copied from the viral DNA sequences acquired during adaptation, they are exact matches to the viral genome and thus serve as excellent guides

crRNA processing

Stages of CRISPR: CRISPR repeats and spacers in the bacterial DNA undergo transcription, the process of copying DNA into RNA (ribonucleic acid). Unlike the double-chain helix structure of DNA, the resulting RNA is a single-chain molecule. This RNA chain is cut into short pieces called CRISPR RNAs.

spacer acquisition

Stages of CRISPR: DNA from an invading virus is processed into short segments that are inserted into the CRISPR sequence as new spacers

The Ames test for mutagenesis relies on a mutant bacterial strain that is defective in hisG

The Ames test for mutagenesis relies on a mutant bacterial strain that is _____ in _____

-transform DNA without the use of competence factors (CF).

Transformation in Gram Negative: -transform DNA ______ the use of _______ ________(__).

False

Transformation in Gram Negative: are NOT sequence specific ... True or False

do not use transformasomes (require channel through outer membrane)

Transformation in Gram Negative: do not use what? what do they use instead?

they are always competent or they become competent when starved

Transformation in Gram Negative: they are______ competent or they become competent when _______

transformasome complex

Transformation in Gram Positive: -involves growth phase-dependent assembly of a ___________ _________ across the cell membrane

-one the transformasome is assembled, the cell is competent, meaning it can import free DNA fragments and incorporate them into its genome by recombination

Transformation in Gram Positive: -one the transformasome is assembled, the cell is ___________, meaning it can import free DNA fragments and incorporate them into its ________ by ________

begins with the synthesis of a signaling molecule (competence factor, CF) and concludes with the import of a single-stranded DNA strand through a transformasome complex

Transformation in Gram Positive: begins with the synthesis of a _____ _____ (competence factor, CF) and concludes with the import of a ____ stranded DNA strand through a _______ ______

the complex is composed of binding protein that captures extracellular DNA floating in the environment, plus proteins that form a transmembrane pore

Transformation in Gram Positive: transformasome complex is composed of _____ ________ that captures extracellular DNA floating in the environment, plus proteins that form a transmembrane ______

1. Generalized recombination 2. Site-specific recombination

Two different DNA molecules in a cell can recombine by one of two main mechanisms:

1. Tautomeric shifts of DNA 2. Oxidative deamination of bases 3. Formation of apurinic sites (missing base) 4. Damage caused by reactive oxygen species

What are 4 things that can lead to spontaneous mutations?

Formation of apurinic sites

a base is removed

X-rays and gamma rays

break the DNA

Generalized transduction

can transfer any gene from a donor to a recipient cell

Specialized transduction

can transfer only a few closely linked genes between cells

Point mutation

change in a single base

Nonsense mutation

changes the amino acid sequence to a stop codon

Missense mutation

changes the amino acid sequence to another

Frame-shift mutation

changes the open-reading frame of the gene

Silent mutation

does not change the amino acid sequence

Ultraviolet rays

form pyrimidine dimers which block replication and transcription

causes crown gall disease

Agrobacterium tumefaciens is beneficial for plant breeding: - causes ___ ___ disease

Insertion and Deletion

(addition) and (subtraction) of one or more bases

Intercalators

(insert between bases) - Ex. Acridine dyes

Contains a tumor-inducing plasmid (Ti) that can be transferred via conjugation to plant cells

Agrobacterium tumefaciens is beneficial for plant breeding: Contains a ____-inducing ______ (____) that can be transferred via _______ to plant cells

plant breeding and engineering

Agrobacterium tumefaciens is beneficial for plant breeding: Provides an indispensable tool for plant _____ and _____

Nucleotide excision repair

An endonuclease removes a patch of single-stranded DNA containing certain types of damaged bases, including dimers

Transduction

-process in which bacteriophages carry host DNA from one cell to another -occurs accidentally as an offshoot of the phage life cycle

Transformation

-process of importing free DNA into bacterial cells -provided the first clue that gene exchange can occur in microorganisms -purpose is to acquire genes that might be useful as the environment changes

Conjugation

-the transfer of DNA from one bacterium to another, following cell-to-cell contact -initiated by a special pilus protruding from the donor cell

1. SOS repair 2. NHEJ or nonhomologous end joining

2 types of Error-Prone DNA repair:

1. Generalized Transduction 2. Specialized Transduction

2 types of Transduction

1. x-rays and gamma rays 2. ultraviolet rays

2 types of electromagnetic radiation that causes mutations?

1. chemical agents 2. electromagnetic radiation

2 types of mutagens:

1. Transition 2. Transversion

2 types of point mutation:

Cells with damaged chromosomes use DNA donated by others of the same species to repair their damaged genes

3 probable functions for Generalized Recombination: Cells with damaged ________ use DNA _____ by others of the _____ species to repair their damaged genes

Recombination is part of a "self-improvement" program that samples genes from other organisms for an ability to enhance the competitive fitness of the cell

3 probable functions for Generalized Recombination: Recombination is part of a "_____-____" program that samples genes from other organisms for an ability to enhance the _______ _______of the cell

Recombination probably first evolved as an internal method of DNA repair, useful to fix mutations or restart stalled replication forks (not involving foreign DNA)

3 probable functions for Generalized Recombination: Recombination probably first evolved as an internal method of ____ ______, useful to fix ________ or restart ______ replication forks (not involving foreign DNA).

endonucleases

3 types of restriction ______

Types I and III

3 types of restriction endonucleases: have their restriction and modification activities combined in one multifunctional protein. And cleave DNA some distance away from the recognition site

Type II

3 types of restriction endonucleases: possesses only endonuclease activity. Generally recognize palindromic DNA sequences and cleave at those sites

1. Methyl mismatch repair 2. photoreactivation 3. nucleotide excision repair 4. base excision repair 5. recombinational repair

5 types of Error-proof repair pathways:

cell function

A number of studies have now shown that CRISPR loci contribute to ____ _____ beyond providing immunity from phage infections.

mutation

is a heritable change in the DNA

Base analogs

mimic bases (ex.) caffeine

reversion mutations colonies

next we add a a chemical that is a mutagent so this mutagen causes ____ mutations and ____ will appear

no growth bc Histidine is important for growth

place the mutant bacterial strain on a medium lacking histidine.. what happens?

Transversion

purine (A/G) ↔ pyrimidine (T/C)

Transition

purine → purine; pyrimidine → pyrimidine

Oxidative deamination

removal of amino group from a base causes it to change to a different base

Site-specific recombination

require a short sequence recognized by the recombination enzyme

Generalized recombination

requires that the two recombining molecules have a considerable stretch of homologous DNA sequences

Site-specific recombination

requires very little sequence homology between the recombining DNA molecules

Enzymatic cleavage (restriction) of alien DNA, by restriction endonucleases

restriction and modification, involves: 1. ________ ________ (restriction) of alien DNA, by ______ _______

Protective methylation (modification) of host DNA

restriction and modification, involves: 2. Protective ______ (modification) of _____ DNA

Tautomeric Shifts

shifts in DNA bases alter base pairing properties (evil twins)

some bacteria are naturally competent and some require manipulations (

some bacteria are _______ competent and some require ________

Restriction and Modification Protection System

there is a protection system in place since there are dangers to the cell associated with the indiscriminate transfer of DNA between bacteria what is it called?

(clustered regularly interspaced short palindromic repeats

what does CRISPR stand for?

Ames test

widely employed method that uses bacteria to test whether a given chemical can cause mutations in the DNA of the test organism