1. Replication, transcription, translation and expression of nucleic acid

2. Central dogma of molecular biology
translation
transcription
replication
DNA
RNA
PROTEIN
Solid arrow indicate types of information transfers that occur in cells. DNA directs its own replication to produce new DNA molecule; DNA is transcribes into RNA; RNA is translated into protein. The dashed lines represent information transfers that occur in certain organisms.

3. Information Flow Replication: DNA duplicates itself
DNA RNA Protein
Transcription: RNA is made on a DNA
template
Translation: Protein is synthesized
from AAs and the three RNAs.
RNA replication: RNA replicates itself
Reverse Transcription: RNA directs
synthesis of DNA
Replication: DNA duplicates itself

4. DNA replication is an anabolic polymerization process, that allows a cell to pass copies of its genome to its descendants.
The key to DNA replication is the complementary structure of the two strands:
Adenine and guanine in one strand bond with thymine and cytosine, respectively, in the other.
DNA replication is a simple concept - a cell separates the two original strands and uses each as a template for the synthesis of a new complementary strand.
Biologists say that DNA replication is semiconservative because each daughter DNA molecule is composed of one original strand and one new strand.

5. Processes in DNA Replication

6. Initial Processes in DNA Replication
DNA replication begins at a specific sequence of nucleotides called an origin.
First, a cell removes chromosomal proteins, exposing the DNA helix.
Next, an enzyme called DNA helicase locally "unzips" the DNA molecule by breaking the hydrogen bonds between complementary nucleotide bases, which exposes the bases in a replication fork. Other protein molecules stabilize the single strands so that they do not rejoin while replication proceeds
After helicase untwists and separates the strands, a molecule of an enzyme called DNA polymerase III binds to each strand.
DNA polymerases replicate DNA in only one direction - 5' to 3' - like a jeweler stringing pearls to make a necklace, adding them one at a time, always moving from one end of the string to the other.
Because the two original (template) strands are antiparallel cells synthesize new strands in two different ways. One new strand, called the leading strand, is synthesized continuously as a single long chain of nucleotides.
The other new strand, called the lagging strand, is synthesized in short segments that are later joined.

7. Synthesis of the Leading Strand
A cell synthesizes a leading strand toward the replication fork in the following series of five steps
1) An enzyme called primase synthesizes a short RNA molecule that is complementary to the template DNA strand. This RNA primer provides the 3' hydroxyl group required by DNA polymerase.
2) Triphosphate deoxyribonucleotides form hydrogen bonds with their complements in the parental strand. Adenine nucleotides bind to thymine nucleotides, and guanine nucleotides bind to cytosine nucleotides.
3) Using the energy in the high-energy bonds of the triphosphate deoxyribonucleotides, DNA polymerase III covalently joins them one at a time by dehydration synthesis to the leading strand.

8. 4) DNA polymerase III also performs a proofreading function
4) DNA polymerase III also performs a proofreading function. About one out of every 100,000 nucleotides is mismatched with its template; for instance, a guanine might become incorrectly paired with a thymine.
DNA polymerase III recognizes most such errors and removes the incorrect nucleotides before proceeding with synthesis. This role, known as the proofreading exonuclease function, acts like the delete key on a keyboard, removing the most recent error.
Because of this proofreading exonuclease function, only about one error remains for every ten billion (1010) base pairs replicated.
5) Another DNA polymerase - DNA polymerase I - ­replaces the RNA primer with DNA. Note that researchers named DNA polymerase enzymes in the order of their discovery, not the order of their actions.

9. Synthesis of the Lagging Strand
The steps in the synthesis of a lagging strand are as follows
As with the leading strand, primase synthesizes RNA primers.
Nucleotides pair up with their complements in the template-adenine with thymine, and cytosine with guanine.
DNA polymerase III joins neighboring nucleotides and proofreads. In contrast to synthesis of the leading strand, however, the lagging strand is synthesized in discontinuous segments called Okazaki fragments. Each Okazaki fragment requires a new RNA primer and consists of 1000 to 2000 nucleotides.
DNA polymerase I replaces the RNA primers of Okazaki fragments with DNA and further proofreads the daughter strand.
DNA ligase seals the gaps between adjacent Okazaki fragments to form a continuous DNA strand.

10. TRANSCRIPTION Cells transcribe four main types of RNA from DNA
RNA primer molecules for DNA polymerase to use during DNA replication
messenger RNA (mRNA) molecules, which carry genetic information from chromosomes to ribosomes
ribosomal RNA (rRNA) molecules, which combine with ribosomal polypeptides to form ribosomes-the organelles that synthesize polypeptides
transfer RNA (tRNA) molecules, which deliver amino acids to the ribosomes

11. Initiation of Transcription
RNA polymerases - the enzymes that synthesize RNA ­bind to specific nucleotide sequences called promoters, each of which is located near the beginning of a gene and initiates transcription.

12. Initiation of Transcription
In bacteria, a polypeptide subunit of RNA polymerase called the sigma factor is necessary for recognition of a promoter.
Once it adheres to a promoter sequence, RNA polymerase unwinds and unzips the DNA molecule in the promoter region and then travels along the DNA, unzipping the double helix as it moves.
One type of RNA polymerase transcribes RNA primer, and a second type of RNA polymerase transcribes mRNA, rRNA, and tRNA.
A cell uses different sigma factors and different promoter sequences to provide some control over the relative amount of transcription.
RNA polymerases with different sigma factors do not adhere equally strongly to all promoters; there is about a 100-fold difference between the strongest attraction and weakest one. The greater the attraction between a particular sigma factor and a promoter, the more likely that transcription will proceed. Ultimately, variations in sigma factors and promoters affect the amounts and kinds of polypeptides produced.

13. Elongation of the RNA Transcript

14. Elongation of the RNA Transcript
Like DNA polymerase, RNA polymerase links nucleotides in the 5' to 3' direction only; however, RNA polymerase differs from DNA polymerase in the following ways:
RNA polymerase unwinds and opens DNA by itself; helicase is not required.
RNA polymerase does not need a primer.
RNA polymerase is slower than DNA polymerase, proceeding at a rate of about 50 nucleotides per second.
RNA polymerase incorporates ribonucleotides instead of deoxyribonucleotides.
Uracil nucleotides are incorporated instead of thymine nucleotides.
The proofreading function of RNA polymerase is less efficient, leaving a base pair error about every 10,000 nucleotides.

15. Termination of Transcription

16. Termination of Transcription: Self-Termination
Self-termination occurs when RNA polymerase transcribes a terminator sequence of DNA composed of two symmetrical series:
one that is very rich in guanine and cytosine bases, followed by a region rich in adenine bases.
RNA polymerase slows down during transcription of the GC rich portion of the terminator because the three hydrogen bonds between each guanine and cytosine base pair make unwinding the DNA helix more difficult.
This pause in transcription, which lasts about 60 seconds, provides enough time for the RNA molecule to form hydrogen bonds between its own symmetrical sequences, forming a stem and loop structure that puts tension on the union of RNA polymerase and the DNA.
When RNA polymerase transcribes the adenine-rich portion of the terminator, the relatively few hydrogen bonds between the adenine bases of DNA and the uracil bases of RNA cannot withstand the tension, and the RNA transcript breaks away from the DNA, releasing RNA polymerase.

17. Termination of Transcription: Rho-Dependent Termination
The second type of termination depends on a termination protein, called Rho, that binds to a specific RNA sequence near the end of an RNA transcript.
The protein moves toward the 3' end, pushing between RNA polymerase and the DNA strand and forcing them apart; this releases RNA polymerase and the RNA transcript.

18. FINAL EXAM

19. 6 ASSAY QUESTIONS
ANSWER 5 QUESTIONS
PART 1 – COMPULSORY
3 QUESTIONS
PART 2 – ANSWER ANY 2

20. TRANSLATION
Translation is the process whereby ribosomes use the genetic information of nucleotide sequences to synthesize polypeptides composed of specific amino acid sequences.

21. How do ribosomes interpret the nucleotide sequence of mRNA to determine the correct order in which to assemble amino acids?

22. The Genetic Code
Genes are composed of sequences of three nucleotides that specify amino acids.
For example, the DNA nucleotide sequence TTT specifies the amino acid lysine, and TTA codes for asparagine.

24. 5’ end Middle base ’ end
U C A G
U phe ser tyr cys U
phe ser tyr cys C
leu ser end end A
leu ser end trp G
C leu pro his arg U
leu pro his arg C leu pro gln arg A
leu pro gln arg G

25. 1. pro 2. arg 3. ser Code Quiz Click for the answer.
1. CCU codes for: ?
2. CGA codes for: ?
3. UCA codes for: ? 5’ U C A G 3’
Phe
Ser
Tyr
Cys
Leu
End
Trp
Pro
His
Arg
Gln
Ile
Thr
Asn
1. pro
2. arg
3. ser

26. 64 possible arrangements - more than enough to specify 21 amino acids.
the genetic code define as triplets of mRNA nucleotides called codons that code for specific amino acids.
61 codons specify amino acids and 3 codons
-UAA, UAG, and UGA-to stop translating
UGA codes for the 21st amino acid, selenocysteine.
Codon AUG also has a dual function, acting as both a start signal and coding for an amino acid – methionine.

27. Some exceptions of the genetic code
Codon
Usual use
Alternative use
AUA
Codes for isoleucine
Codes for methionine in mitochondria
UAG
STOP
Codes for glutamine in some protozoa and algae and for pyrrolysine (22nd amino acid) in some prokaryotes
CGG
Codes for arginine
Codes for tryptophan in plant mitochondria
UGA
STOP, selenocysteine
Codes for tryptophan in mitochondria and mycoplasmas (type of bacteria)

28. Participants in Translation: Messenger RNA
Messenger RNA carries genetic information (in the form of RNA nucleotide sequences) from a chromosome to ribosomes.
In prokaryotes a basic mRNA molecule contains sequences of nucleotides that are recognized by ribosomes:
an AUG start codon, sequential codons for other amino acids in the polypeptide, and at least one of the three stop codons. A single molecule of prokaryotic mRNA often contains a start codon and instructions for more than one polypeptide arranged in series.
Because both transcription and the subsequent events of translation occur in the cytosol of prokaryotes, prokaryotic ribosomes can begin translation before transcription is finished.

30. Participants in Translation: Transfer RNA
tRNA molecule is a sequence of about 75 ribonucleotides that curves back on itself to form three main hairpin loops (a)
For simplicity, tRNA will be represented in subsequent figures by an icon shaped like the illustration (b)

31. A molecule of tRNA transfers the correct amino acid to a ribosome during polypeptide synthesis. To this end, tRNA has an acceptor stem for a specific amino acid at its 3' end, and an anticodon triplet in its bottom loop.
The existence of only one specific charging enzyme for each amino acid ensures that every tRNA molecule carries only one specific amino acid.
Anticodons are complementary to mRNA codons, and each acceptor stem is designed to carry one particular amino acid, which varies with the tRNA.

32. Participants in Translation: Ribosomes and ribosomal RNA

33. Prokaryotic ribosomes, which are also called 70S ribosomes based on their sedimentation rate in an ultracentrifuge, are extremely complex associations of ribosomal RNAs and polypeptides. Each ribosome is composed of two subunits: 50S and 30S.
The 50S subunit is in turn composed of two rRNA molecules (23S and 5S) and about 34 different polypeptides, whereas the 30S subunit consists of one molecule of 165 rRNA and 21 ribosomal polypeptides. The ribosomes of mitochondria and chloroplasts are also 70S ribosomes composed of the similar subunits and polypeptides.
In contrast, both the cytosol and the rough endoplasmic reticulum (RER) of eukaryotic cells have 80S ribosomes composed of 60S and 40S subunits. These subunits contain larger molecules of rRNA and more polypeptides than the corresponding prokaryotic subunits, though researchers do not agree on the exact number of polypeptides. The term eukaryotic ribosome is understood to mean only the 80S ribosomes of the cytosol and RER. Since the ribosomes of mitochondria and chloroplasts are 70S, they are called prokaryotic ribosomes even though they are in eukaryotic cells.

34. Each ribosome also has three tRNA binding sites that are named for their function:
1) The A site accommodates a tRNA delivering an amino acid.
2) The P site holds a tRNA and the growing polypeptide.
3) Discharged tRNAs exit from the E site.

35. Stages of Translation Initiation: the events
1) The smaller ribosomal subunit attaches to mRNA at a ribosome binding site (also known as a Shine-Dalganno sequence after its discoverers), with a start codon at its P site.

36. 2) tRNA £Met (whose anticodon is complementary to the start codon) attaches at the ribosome's P site; GTP supplies the energy required for binding.

37. 3) The larger ribosomal subunit attaches to form a complete initiation complex.

38. Stages of Translation Elongation
1) The transfer RNA whose anticodon matches the next codon - in this case, phenylalanine (Phe) - delivers its amino acid to the A site. Another protein called elongation factor escorts the tRNA along with a molecule of GTP. Energy from GTP is used to stabilize each tRNA as it is added to the A site.

39. 2) A ribozyme in the larger ribosomal subunit forms a peptide bond by dehydration synthesis between the terminal amino acid of the growing polypeptide chain (in this case, N-formylmethionine) and the newly intro­duced amino acid. The polypeptide is now attached to the tRNA occupying the A site.
3) Using energy supplied by more GTP, the ribosome moves one codon down the mRNA. This transfers each tRNA to the adjacent binding site; that is, the first tRNA moves from the P site to the E site, and the sec­ond tRNA (with the attached polypeptide) moves to vacated P site.

40. 4) The ribosome releases the “empty" tRNA from the E site
4) The ribosome releases the “empty" tRNA from the E site. In the cytosol, the appropriate enzyme recharges it with another molecule of its specific amino acid.
5) The cycle repeats, each time adding another amino acid (in this case, threonine, then alanine, and then glutamine).

41. Stages of Translation Termination
Termination does not involve tRNA; instead, proteins called release factors halt elongation.
It appears that release factors somehow recognize stop codons and modify the larger ribosomal subunit in such a way as to activate another of its ribozymes, which severs the polypeptide from the final tRNA (resident at the P site). The ribosome then dissociates into its subunits.
Termination of translation should not be confused with termination of transcription. The polypeptides released at termination may function alone as proteins, or they may function together in quarternary protein structures.

42. Regulation of Genetic Expression
About 75% of genes are expressed at all times; that is, they are constantly transcribed and translated and play a persistent role in the phenotype.
These genes code for RNAs and polypeptides that are needed in large amounts by the cell­ for example, integral proteins of the cytoplasmic membrane, structural proteins of ribosomes, and enzymes of glycolysis.
Other genes are regulated so that the polypeptides they encode are synthesized only when a cell has need of them. Protein synthesis requires a large amount of energy, which can be conserved if a cell forgoes production of unneeded polypeptides.
Cells regulate protein synthesis in many ways. They may stop translation directly or stop polypeptide synthesis by stopping mRNA transcription.

43. Control of Translation
Some regulation of genetic expression is at the level of translation; that is, cells control which mRNA molecules are translated into polypeptides.
One way a cell establishes control involves so-called riboswitches. A riboswitch is a molecule of mRNA that changes its shape in response to an alteration in temperature or a shift in the concentration of a nutrient, such as a vitamin, nucleotide base, or amino acid.
Riboswitches fold in such a way as to block ribosomes and translation of the polypeptide they encode when that polypeptide is not needed by the cell.

44. Another method of translational control involves short interference RNA (siRNA), which is an RNA molecule complementary to a portion of mRNA, tRNA, or a gene.
Such RNA molecules are also called antisense RNA. siRNA binds to its complementary nucleic acid, rendering its target inactive.

45. OPERON
An operon consists of a promoter and a series of genes, which code for enzymes and structures such as channel proteins.
Some operons are controlled by an adjacent regulatory element called an operator where a repressor protein binds to stop transcription.
Such operons are either repressed (turned off) or induced (turned on) by proteins coded by a regulatory gene (located elsewhere).
Inducible operons are not usually transcribed and must be activated by inducers.
Repressible operons operate in reverse fashion-they are transcribed continually until deactivated by repressors.

46. The Lactose Operon, an Inducible Operon
The lactose (lac) operon is inducible operon. It includes a promoter, an operator, and three genes that encode for protein involved in the catabolism of lactose.
The operon is controlled by a regulatory gene that is constantly transcribed and translated to produce a repressor protein that attaches to DNA at the lac operator.
This repressor prevents RNA polymerase from moving be­yond the promoter, stopping synthesis of mRNA. Thus, the lac operon is usually inactive.

47. Whenever lactose becomes available, the cell takes in lactose and converts it to allolactose - an inducer that changes the quaternary structure of the repressor so that it is inactivated and can no longer attach to DNA.
This absence of binding al­lows transcription of the three structural genes to proceed­the operon has been induced and has become active. Ribosomes translate the newly synthesized mRNA to produce enzymes that catabolize lactose.
Once the lactose supply has been depleted, there is no more inducer, and the repressor once again becomes active, suppressing transcrip­tion and translation of the lac operon. In this manner, its conserve energy by synthesizing enzymes for the ca­tabolism of lactose only when lactose is available to them.
Such inducible operons are often involved in controlling catabolic pathways whose polypeptides are not needed

48. The Tryptophan Operon, a Repressible Operon

49. The tryptophan (trp) operon, which consists of a promoter, an operator, and five genes that code for the enzymes involved in the synthesis of tryptophan, is an example of such a repressible operon.
Just as with the lac operon a regulatory gene codes for a repressor molecule that is constantly synthesized. In contrast to inducible operons however, the repressor of repressible operons is normally inactive.
In the case of the repressible trp operon whenever tryptophan is not present in the environment, the trp operon inactive:
The appropriate mRNA is transcribed the enzymes for tryptophan synthesis are translated, and tryptophan is produced (Figure a).
When tryptophan is available, it activates the repressor by binding to it. The activated repressor then binds to the operator, halting the movement of RNA polymerase and halting transcription (Figure b).
In other words, tryptophan acts as a corepressor of its own synthesis.

50. The roles of operons in the regulation of transcription
Type of regulation
Type of metabolic pathway regulated
Regulating condition
Inducible operons
Catabolic pathways
Presence of substrate of pathway
Repressible operons
Anabolic pathways
Presence of product of pathway

51. Mutations of Genes: Types of mutation
Mutations range from large changes in an organism's genome, such as the loss or gain of an entire chromosome, to the most common type of mutation - point mutations - in which just one nucleotide base pair is affected.
Mutations include base pair insertions, deletions, and substitutions.
Substitution of a nucleotide of similar shape - a purine for a purine or pyrimidine for a pyrimidine-is called a transition.
Substitution of a purine for a pyrimidine or vice versa is called a transversion.

52. The following analogy illustrates some types of mutations.
Suppose that the DNA code was represented by the letters
THECATATEELK.
Grouping the letters into triplets (like codons) yields
THE CAT ATE ELK.
The substitution of a single letter could either change the meaning of the sentence, as in THE RAT ATE ELK, or result in a meaningless phrase, such as THE CAT RTE ELK.
Insertion or deletion of a letter produces more serious changes, such as
TRH ECA TAT EEL K or TEC ATA TEE LK.
Insertions and deletions are also called frame shift mutations because nucleotide triplets subsequent to the mutation are displaced, creating new sequences of codons that result in vastly altered polypeptide sequences.
Frameshift mutations affect proteins much more seriously than mere substitutions because a frame shift affects all co dons subsequent to the mutation.
Mutations can also involve inversion (THE ACT ATE KLE),
duplication (THE CAT CAT ATE ELK ELK), or
transposition (THE ELK ATE CAT).
Such mutations and even larger deletions and insertions are gross mutations.

53. Effects of Mutations
Some base-pair substitutions produce silent mutations because the substitution does not change the amino acid sequence because of the redundancy of the genetic code.
For example, when the DNA triplet AAA " is changed to AAG, the mRNA codon will be changed from UUU to UUC; however, because both codons specify the amino acid phenylalanine, there is no change in the phenotype - the mutation is silent because it affects the genotype only.

54. Of greater concern are substitutions that change a codon for one amino acid into a codon for a different amino acid.
A change in a nucleotide sequence resulting in a codon that specifies a different amino acid is called a missense mutation; what gets transcribed and translated makes sense, but not the right sense.
The effect of missense mutations depends on where in the protein the different amino acid occurs.
When the different amino is in a critical region of a protein, the protein becomes nonfunctional; however, when the different amino acid is in a less important region, the mutation has no adverse effect.

55. A third type of mutation occurs when a base-pair substitution changes an amino acid codon into a stop codon.
This is called a nonsense mutation. Nearly all nonsense mutations result in nonfunctional proteins.

56. Frameshift mutations (that is, insertions or deletions) typically result in drastic missense and nonsense mutations, except when the insertion or deletion is very close to the end of a gene

57. Mutagens
Mutations occur naturally during the life of an organism. Such spontaneous mutations result from errors in replication and repair as well as from recombination in which relatively long stretches of DNA move among chromosomes, plasmids, and viruses, introducing frame shift mutations.
Further, though cells have repair mechanisms to reduce the effect of mutations, the repair process itself can introduce additional errors.
Physical or chemical agents called mutagens, which include radiation and several types of DNA-altering chemicals, induce mutations.

58. Radiation
Ioning radiation, such as X rays and gamma rays, can cause some of the molecules within cells to lose electrons, becoming highly reactive ions and free radicals.
Some of these reactive ions and free radicals can combine with bases on DNA, resulting in errors in DNA replication and therefore mutations. Even more seriously, these groups can react with the sugar-phosphate backbone of DNA, causing breaks in chromosomes.
Nonionizing radiation in the form of UV light is also mutagenic because it can cause adjacent thymine bases to covalently bind to another, producing thymine dimmers.
Such dimmers can cause serious harm or death to a cell if they are not repaired, since these dimmers prevent the cell from properly transcribing or replicating such DNA.

59. Chemical Mutagens : Nucleotide Analogs
nucleotide analogs are compounds that are structurally similar to normal nitrogenous bases, but with different base-pairing properties.
These compounds can become incorporated into growing DNA during replication, replacing their related base.
Once incorporated, the nucleoside analog can inhibit further replication, or cause mismatching in a future round of replication.
For example, 5-bromouracil is a nucleoside analog of thymine, but it often pairs with guanine rather than adenine. Incorporation of 5-bromouracil can therefore lead to a base substitution mutation of a guanine for an adenine.

60. Chemical Mutagens : Nucleotide-Altering Chemicals
some chemical mutagens directly alter the structures of the nitrogenous bases of DNA.
For example, nitrous acid can chemically alter adenine bases so that they base pair with cytosine, rather than thymine.
During replication, this change causes base substitution mutations in the daughter DNA.

61. Chemical Mutagens : Frameshift Mutagens
some chemical mutagens cause small insertions or deletions of nucleotide base pairs, which can lead to frameshift mutations.
Examples of such frameshift mutagens include acridine, a dye commonly used as a mutagen in genetic research

62. Frequency of Mutation Mutations are rare events.
Organisms could not live or effectively reproduce themselves. About one of every ten million (107) genes contains an error.
Mutagens typically increase the mutation rate by a factor of times; mutagens induce an error in one of every 104 to 106 genes.
Most mutations are deleterious because they code for nonfunctional proteins or stop transcription entirely. Cells, without functional proteins cannot metabolize; any deleterious mutations are removed from the population when the cells die.
Rarely, a cell acquires a beneficial mutation that allows it to survive, reproduce, and pass the mutation to its descendants.
For example, a bacterium night randomly acquire a mutation that confers resistance to an antibiotic.

63. DNA Repair
Although a mutation might rarely convey an advantage, most mutations are deleterious.
Methods for repairing damaged DNA, including light and dark repair of pyrimidine dimers, base-excision repair, mis­match repair, and an S0S response.

64. Repair of Pyrimidine Dimers
Many cells contain DNA photolyase, an enzyme that is activated by visible light to break the bonds between adjoining pyrimidine nucleotides, reversing the mutation and restoring the original DNA sequence.
Light repair mechanism is advantageous for the prokaryote, but it presents a difficulty to scientists studying UV-induced mutations-­they must keep such strains in the dark, or the mutants revert to their previous form.
Dark repair involves a different repair enzyme-one that doesn't require light. Dark repair enzymes cut the damaged section of DNA from the molecule, creating a gap that is repaired by DNA polymerase I and DNA ligase. Dark repair operates either in light or in the dark.

65. Base-Excision Repair
Sometimes DNA polymerase III incorporates an incorrect nucleotide during DNA replication. If the proofreading function of DNA polymerase III does not repair the error, cells may use another enzyme system in a process called base-excision repair.
This enzyme system excises the erroneous base, and then DNA polymerase I fills in the gap

66. Mismatch Repair A similar repair mechanism is called mismatch repair.
Mismatch repair enzymes scan newly synthesized DNA looking for mismatched bases, which they remove and replace.
How does the mismatch repair system determine which strand to repair? If it chose randomly, 50% of the time it would choose the wrong strand and introduce mutations.
Mismatch repair enzymes, however, do not choose randomly.
They distinguish between a new DNA strand and an old strand because old strands are methylated.
Recognition of an error as far as 1000 base pairs away from an unmethylated portion of DNA triggers the mismatch repair enzymes. Once a new DNA strand is methylated, mismatch repair enzymes cannot correct any errors that remain.

67. SOS Response
Sometimes damage to DNA is so extreme that regular repair mechanisms cannot cope with the damage.
In such cases, bacteria resort to what geneticists call an S0S response involving a variety of processes, such as the production of novel DNA polymerases (IV and V) capable of copying less-than-perfect DNA.
These polymerases replicate DNA with little regard to the base sequence of the template strand.
Of course, this introduces many new and potentially fatal mutations, but presumably SOS repair allows a few offspring of these bacteria to survive.