Repair/Recommbination and Transcription in Prokaryotes
Mismatch repair in E. coli
- A G-T mismatch is recognized by MutS - MutL binds to MutS and activates the MutH nuclease - MutH cleaves the backbone in the vicinity of the mismatch - A segment of the DNA strand containing the erroneous T is removed by exonuclease I and synthesized anew by DNA polymerase III
required substrates of RNA polymerase
- DNA - ribonucleoside triphosphates: ATP, GTP, UTP, & CTP
Components of RNA polymerase
- a template - activated precursors - a divalent metal ion: either Mn2+ or Mg2+
How does allolactose and cAMP combine to regulate the lac operon
- allolactose binds to the repressor protein, preventing it from binding to the operator and allowing the polymerase access to the gene - cAMP binds to CAP; this complex binds the lac operon upstream of the promoter and stimulates the binding of polymerase to the promoter
nucleotide excision repair
- an enzyme complex recognizes the distortion of the DNA caused by the offending base - the UvrABC excinuclease cleaves the DNA at two sites, several nucleotides on each side of the distortion - DNA polymerase I and DNA ligase close the resulting gap
what inhibits transcription of RNA
- antibiotics rifampicin - actinomycin
Protein Rho
- binds to a particular sequence on the RNA product and uses the energy of ATP hydrolysis to chase down the polymerase in the transcription bubble - causes the transcription bubble to dissociate
How do double-strand breaks happen?
- breaks arise when replication stalls, such as when the polymerase encounters an unpaired nick in one the template strands at the replication fork; the replication fork collapses, leaving a double strand break on one of the daughter helices - breaks can also result from exposure to ionizing radiation
How does the sigma subunit recognize a promoter site?
- by decreasing the affinity of the polymerase for DNA, the sigma subunit allows the enzymes to rapidly scan the DNA for a promoter - this decrease allows the enzyme to bind to the DNA and slide along it, searching for the promoter - once the promoter is located and RNA synthesis begins, the sigma subunit dissociates from the enzyme to assit another polymerase in initiation
Why is the most common point mutation in humans C --> T?
- cytosine in eukaryotic DNA is frequently methylated in the 5th position - when 5-methylcytosine spontaneously delaminates, thymine is formed
Transcription in E. coli
- direction of RNA synthesis is 5' to 3' - the nucleoside triphosphates add to the 3' end of the growing RNA strand - the growing chain is complementary to the template DNA strand - catalyzed by RNA polymerase - the synthesis is driven forward by the hydrolysis is pyrophosphate
Catabolite repression
- glucose concentration increases - cAMP concentration falls - the lac operon is not expressed
allolactose
- inducer of the lac operon - upon binding allolactose, the lac repressor undergoes a structural change that reduces its affinity for DNA - RNA polymerase can then transcribe the structural genes of the operon
DNA components of regulatory system
- operator site - promoter - structural gene
biological processes that involve recombination
- repair of DNA - segregation of chromosomes during meiosis - enhancement of genetic diversity
Protein dependent termination (EXTRINSIC)
- requires protein rho
DNA damage
- resulting from errors in replication or environmental insults - may lead to cell death or uncontrolled replication, as when normal cells are transformed into cancer cells
enlongation
- takes place after the DNA is unwound - the transcription bubble moves along the DNA as DNA is unwound and then rewound, and the RNA product is extruded from the complex
recombination process for repair of double-strand break
- the double-strand break is recognized, and the 5' ends are digested, generating single-strand regions that are bound by RAD51 - strand invasion occurs when a single strand from the damaged DNA replaces a strand in the undamaged DNA; the resulting three-stranded structure is caed a displacement loop - DNA synthesis occurs using the undamaged DNA as a template - a second strand invasion occurs to complete the repair, forming a structure called a Holiday junction - cleavage and ligation yield two intact helices
termination phase
- the formation of phosphodiester linkages stops - the RNA-DNA hybrid dissociates - the melted region of DNA reanneals - RNA polymerase releases the DNA
Uracil repair
- use of thymine instead instead of uracil in DNA allows the detection of deamination of cytosine - If uracil is detected in DNA, it is removed by uracil DNA glycosylase and the resulting AP is repaired with the insertion of cytosine
DNA sequences that act as a promoter for many bacteria
-10 sequence (Pribnow box) and the -35 sequence
B-galactosidase
-breaks down lactose into galactose and glucose - generates allolactose in a side reaction
termination of rna synthesis
-stop signal - intrinsic termination
catabolite activator protein (CAP)
1. cyclic AMP binds to CAP 2. cAMP-CAP complex binds to DNA near start of lac operon 3. stimulates activity of RNA polymerase
stages of RNA synthesis
1. initiation 2. elongation 3. termination
ribosomal and transfer rna modifications
1. mature RNA cleaved from large precursor 2. transfer RNA transcript lack the CCA sequence at 3' end; these nucleotides are added post transcriptionally 3. bases and ribose of tRNA and rRNA are modified by attachment of methyl groups.
Outline of DNA repair systems
1. recognize the inappropriate base(s) 2. remove the inappropriate base(s) 3. repair the resulting gap with a DNA polymerase and DNA ligase
How many strands of DNA are present in a Holliday junction?
4 strands
What would happen if uracil naturally occurred as a base in DNA?
Cytosine spontaneously deaminates to form uracil. If uracil naturally occured as a base in DNA, cytosine deamination would lead an A-U pair replacing a G-C pair after the next round of replication
intermediate in rna synthesis
DNA + RNA hybrid helix 8 nucleotides
What property of the structure of DNA allows for the repair of bases damaged by mutation?
DNA's property of being a double helix. Damage to one strand can be repaired by the use of the other strand as a template
transcription
DNA-directed synthesis of RNA catalyzed by RNA polymerase
Mismatch repair proteins in E. coli
MutS, MutL, MutH - MutH is the endonuclease
intrinsic termination
RNA forms hairpin (has a stem and a loop) + 4 or more uracil residues 1. the RNA product that is weakly bound because of the U-A base pairs (unstable pair), is released 2. DNA double helix reforms
How does base excision repair work?
The DNA backbone at the AP site is cleaved by AP endonuclease, and a phosphodiesterase excises the deoxyribose phosphate. The gap is repaired by DNA polymerase I and DNA ligase
sigma (o~) subunit
a component of bacterial RNA polymerase that enables the core RNA polymerase to recognize promoter sites
Holiday junctions
a crosslike structure, formed by four polynucleotide chains, that is a key intermediate in the recombination process
tumor suppressor genes
a gene that encodes a protein, such as a DNA-repair protein, that suppresses tumor development when at least one copy of the gene is free of a deleterious mutation
RAD51
a key ATPase in human recombination that binds single-stranded DNA - the double-strand break is recognized, and the 5' ends are digested, generating single-strand regions that are bound by RAD51
base excision repair
a means of repairing DNA in which the damaged base is removed and replaced by a base complementary to the undamaged DNA strand - the enzyme AlkA removes the base - the site of the missing base is called an AP site
transcription factors
a protein that assists RNA polymerase in the initiation of RNA synthesis; binds to a specific promoter element
Adenine deamination
adenine can be deaminated, forming hypoxanthine, which pairs with cytosine instead of thymine
Alkylation example
aflatoxin B1 is converted into a highly reactive epoxide that reacts with guanine, forming a compound that, during replication, converts a G-C base pair into an A-T base pair
micro RNA
appears to regulate the expression of genes, possibly via binding to specific nucleotide sequences
upstream element
bound by the alpha subunit of RNA polymerase and increases the efficiency of transcription by creating an additional binding site for the polymerase
translesion polymerases
can replicate across the damage and generate a rough draft of the damaged sequence that can be at least partly repaired by DNA repair processes
What does RNA polymerase catalyze?
catalyzes the initiation & elongation of RNA strands - RNA polymerase does not require a primer
mutagens
chemicals that alter specific bases after replication is complete - perturbs the base sequence of DNA and causes a mutation - can also be energy sources such as ultraviolet light
holoenzyme of RNA polymerase
consisting of a2BB'wo~ subunits, initiates RNA synthesis
mismatch repair
corrects errors not corrected by proofreading - system consists of at least 2 proteins, one for detecting the mismatch and the other for recruiting an endonuclease that cleaves the newly synthesized DNA strand close to the lesion; an exonuclease can then excise the incorrect base, and a polymerase fills the gap
direct repair
corrects mistakes without having to remove any fragments of DNA - ex. pyrimidine dimers are simply cleaved to restore the original nucleotides
high-energy electromagnetic radiation
damage DNA by producing high concentration of reactive chemicals - can cause damage by single- and double-stranded breaks in DNA
Core enzyme of RNA polymerase
elongates the RNA product - composed of 2 alpha subunits, beta, beta prime, and omega
messenger RNA's
encode the amino acid sequence of all the polypeptides found in the cell
Cytochrome P450 enzymes
enzyme that converts some compounds to a highly reactive epoxide - ex. can convert cigarette smoke and automobile exhaust
trinucleotide repeats
found in stretches of DNA in which a trinucleotide sequence is repeated many times - these segments of DNA can expand in the course of DNA replication, causing genetic diseases such as Huntington disease
Rifampicin inhibits:
inhibits initiation by blocking the channel into which the RNA-DNA hybrid generated by the enzyme must pass
how actinomycin inhibits transcription
intercalates between the bases of the DNA double helix, preventing the DNA from being used as a template for RNA synthesis
lac operon
lactose metabolizing enzymesin the presence of lactose, these are expressed - B-galactosidase - galactoside permease - thiogalactoside transacetylase
transfer RNA's
match specific amino acids to triplet codons in mRNA during protein synthesis
alkylation
nucleotide bases are subject to the addition of a hydrocarbon molecule
strand invasion
occurs when a single strand from the damaged DNA replaces a strand in the undamaged DNA - the resulting three-stranded structures
Hydroxyl radicals
oxidize guanine to 8-oxoguanine, which base-pairs with adenine instead of cytosine during the next round of replication
homologous recombination
parent DNA duplexes align at regions of sequence similarity, and new DNA molecules are formed by the breaking and joining of homologous segments
mutagenic
permanent changes in the DNA sequence
the Lac Operon illustrates the control of
prokaryotic gene expression
mRNA strand
same as the coding strand but has U instead of T - ex. 5' - ACUGC - 3'
what does "s" mean
sedimentation rate
promoters
specific DNA sequences that direct RNA polymerase to the proper initiation site - usually just upstream of a gene
closed promoter complex
the DNA in the promoter region is bound to polymerase but not unwound - the complex is formed when the promoter is initially located by the polymerase
coding strand
the coding strand of DNA has the same sequence as that of the RNA transcript - ex. 5' - ACTGC - 3'
Ribosomal RNA
the constituents & catalyze appropriate amino acid addition in ribosomes
What does base excision repair corrects?
the deamination of methyl cytosine to thymine - T in the T-G pair always treated as the incorrect base
recombination
the exchange of genetic information between two DNA molecules - two daughter molecules of DNA are formed by the exchange of genetic material between two parental strands - useful for repair of extensive damage to the DNA, such as breaks in both strands of the DNA
Xeroderma pigmentosum
the human genetic skin disease that is caused by a mutation in components of the human nucleotide-excision-repair pathway
Simplest source of damage in the double helix:
the incorporation of an incorrect base during replication that escapes the notice of the proofreading capabilities of the DNA polymerases
What happens when base excision repair fails?
the mutation may be corrected by the nucleotide excision repair system
transcription bubble
the region containing the RNA polymerase, DNA, and the RNA product
lac operon regulation
the regulatory gene encodes a protein called the lac repressor that binds to the operator site in the absence of lactose and prevents transcription of the structural genes
template strand
the sequence of the template strand of DNA is the complement of that of the RNA transcript - ex. 3' - TGACG - 5'
open promoter complex
the two chains in the AT-rich promoter region are separated - RNA polymerase unwinds approximately 17 bases - after the segment is unwound the stage is set for the formation of the first phosphodiester linkage of the new RNA chain
Why is thymine used in DNA instead of uracil?
to preserve the integrity of the genetic code
Stop Codon in RNA synthesis
transcribed product of palindromic DNA
Effects of ultraviolet on DNA
ultraviolet radiation covalently links adjacent pyrimidines, thereby blocking replication and gene expression
DNA photolyase
uses light energy to cleave pyrimidine dimers
