Strain Development

Repair mechanism

Industrial Biotechnology
Mutant formation

DNA occur in structural change

i. pyrimidine dimers

ii. chemical changes of single bases

iii. crosslinks between the complementary DNA strands

iv. intercalation of mutagenic agents into DNA

v. single-strand breaks

vi. double-strand breaks

Repair mechanisms

• Photoreactivation

• Mismatch

• Excision repair

• SOS repair

• Adaptive repair
Photoreactivation

• DNA repair mechanism involving photolyase is called

photoreactivation

• These enzyme repair the damage caused by exposure to UV light and

require visible light both for the own activation and for the actual DNA
repair

• Recovery of UV irradiated damage of DNA by visible light

• A method by which cells recovers its DNA after UV exposure

Mismatch repair
• Present in essentially all cells to correct errors that are not corrected by
proofreading

• Consist of two proteins, one detects mismatch and other recruits an endonuclease
that cleaves the newly synthesized DNA strand close to the region of damage

• Sometime DNA polymerases incorporates an incorrect nucleotides during strand

synthesis and 3 to 5 editing system, exonuclease, fails to correct it

• These mismatches as well as single base insertions and deletions are repaired by
the mismatch repair mechanisms.

• Mismatch repair relies on a secondary signal within the DNA to distinguish

between the parental strand and daughter strand, which contains the replication
errors.
Excision repair

Mechanisms that remove damage nucleotide and replace it with an

undamaged nucleotide complementary to that found in the undamaged
DNA strand

 Base excision repair

 Nucleotide excision repair

Base excision repair
• Cellular mechanism that repairs damaged DNA throughout the cell cycle

• Responsible for primarly removing small, non-helix-distorting lesions from the

genome

• Repair damage to a single nitrogeneous base by deploying enzymes called

glycosylases which remove a single nitrogeneous base to create an apurinic or
apyrimidinic site (AP site)

• AP endonuclease nick the damaged DNA backbone at the AP site

• DNA polymerase then removes the damaged region using its 5 to 3 exonuclease
activity and correctly synthesizes the new strand using the complementary strand as a
template
Nucleotide excision repair
• A particularly important excision mechanism that removes DNA damage induced by
UV

• Damaged regions are removed in 12-24 nucleotide long strands in a three-step

process which consists of recognition of damaged DNA both upstream and
downstream of damaged endonucleases and resynthesis of removed DNA region

• Recognition of the damage leads to removal of a short single-stranded DNA segment

that contains the lesion

• The undamaged single-stranded DNA remains and DNA polymerase uses it as

complementary sequence.

• A highly evolutionarily conserved repair mechanism and is used in all eukaryotic and
prokaryotic cells
SOS repair

• Occur when cells are overwhelmed by UV damage

• This allows the cell to survive but at the cost of mutagenesis

• Very likely result in mutation

• Response is only triggered when other repair systems fails as

they are overwhelmed by the increased amount of damage so
that unrepaired DNA accumulates in the cell.

• The accumulation of DNA damage leads to repair induction.

Adaptive Repair

• In E.coli – because of sublethal concentration of alkylating agents

( increase resistant to mutagenic and lethal effects of high doses of alkylating

agents occur)

• Further inducible repair system

• Reduce O6- methylguanine in the genome

• O6- methylguanine DNA transferase is induced

• Mechanism is not understood yet

• Repair system has limit

• With larger mutagen – mutation frequency is directly proportional to O 6-

methylguanine concentration
Mutagens or causes of mutations

Radiation (physical mutagen)

Long wavelength (e.g UV-light within 200-300 nm)

Short wavelength (e.g UV-light within 300-400 nm)

Ionizing (e.g Gamma rays, X-rays)

Can interact with compounds in the cell generating free radicals which
causes chemical damage to DNA
Chemical mutagen
Two major classes

1). Alkylating agents

 Can mutate both replicating and non replicating DNA

2). Base analogs

 Can mutate replicating DNA

• Many chemical mutagens, some exogeneous, some man-made, some

environmental are capable of damaging DNA.

• Many chemotherapeutic drugs and intercalating agents drugs function by

damaging DNA.

• e.g NTG
Mutator Genes

IS-element

• DNA sequence of variable length (800-1400 bp) which can

incorporated in different sites of the genome and released again

Transposons

• Jumping gene that can copy and paste or cut and paste in the genome

Bacteriophage MU

• Linear and include 55 genome and transposable element