What is the function of the following enzymes or proteins in DNA replication: pr

Question

What is the function of the following enzymes or proteins in DNA replication: primase, ligase, DNA polymerase 5' - 3' polymerizing activity, DNA polymerase 3' to 5' editing function, DNA polymerase 5' to 3' exonuclease activity, initiator protein, helicase, single stranded DNA binding protein, clamp protein, topoisomerase. Describe what would happen during DNA replication if a mutation occurred in the genes encoding each of these proteins (i.e., how would a mutation in the topoisomerase gene affect DNA replication?)

Explanation / Answer

Primase :

Normal function:

Primase synthesizes a short RNA primer approximately 11 ±1 nucleotides long, to which new nucleotides can be added by DNA polymerase. It binds to the DNA helicase forming a complex called the primosome , a proces which is seen in bacteria.  In E. coli, primase synthesizes around 2000 to 3000 primers at the rate of one primer per second.

It prevents the leading strand from outpacing the lagging strand by halting the progression of the replication fork.

The other functions of primase are  polymerization of DNA or RNA, terminal transfer, translesion synthesis (TLS), non-homologous end joining (NHEJ) . It may also be involved in restarting stalled replication forks.

Effects of mutation:

RNAP domain of DnaG has three subdomains , the central domain consisting of a 5 stranded sheet and 6 helices . The central domain and the N-terminal domain form a shallow cleft, which makes up the active site of the RNA chain elongation in DnaG.

The opening of the cleft is lined by several highly conserved basic residues: Arg146, Arg221, and Lys229.

These residues are part of the electrostatically positive ridge of the N-terminal subdomain. It is this ridge that interacts with the ssDNA and helps guide it into the cleft, which consists of the metal binding center of the toprim motif on the central subdomain, and the conserved primase motifs of the N-terminal domain.

Those residues include Tyr88, Ile119, and Ile125. Tyr88 is close in proximity to, but does not make contact with, the HBD of DnaG. Mutation of Tyr88 inhibits the formation of the N-terminal domain helical bundle of DnaB, interrupting the contacts with the HBD of DnaG.

One other residue that has been identified as playing a crucial role in the interaction of DnaB and DnaG is Glu15. Mutation of Glu15 does not disrupt the formation of the DnaB, DnaG complex, but instead plays a role in modulating the length of primers synthesized by DnaG.

2) Ligase:

DNA ligase is an enzyme that repairs irregularities or breaks in the backbone of double-stranded DNA molecules. It has three general functions: It seals repairs in the DNA, it seals recombination fragments, and it connects Okazaki fragments (small DNA fragments formed during the replication of double-stranded DNA).

Effects of mutations of Ligase:

Cells that have mutated forms of ligase were shown to exhibit retarded joining of Okazaki fragments during DNA replication and hypersensitivity to a variety of DNA-damaging agents.

3) DNA polymerase 5' to 3' polymerizing activity

Function :

DNA polymerase functions by synthesizing DNA from deoxyribonucleotides, the building blocks of DNA .They add free nucleotides only to the 3' end of the newly forming strand which results in elongation of the newly forming strand in a 5'-3' direction.

Effect of mutation :

Mutations can both increase and decrease exonuclease activity and consequently impair DNA ligation. It has been observed that inactivation of the exonuclease activity causes an increase in mtDNA mutations and premature ageing phenotypes in mice. These mutator mice also contain high levels of truncated, linear fragments of mtDNA.

4) DNA polymerase 3' to 5' editing function :

The 3' to 5' activity of DNA polymerase can only remove one mononucleotide at a time .This is called as proof reading .

Effect of mutation :

Mutation on 3' to 5' activity of DNA polymerase could cause variation in the mtation rate of the enzyme .THis is seen in mutation of epsilon gene . Loss of proofreading due to mutations in the DNA polymerase epsilon gene, lead to a hyper-mutated genotype with more than 100 mutations per Mbase of DNA in human colorectal cancers.

5) DNA polymerase 5' to 3' exonuclease activity :

A conformational change between the different dNTPs is necessary for active discrimination by Polymerase I. Once this change has occurred, a check is done by Pol for proper geometry and proper alignment of the base pair, formed between bound dNTP and a matching base on the template strand. Therefore, the correct geometry of A=T and GC base pairs are the only ones that can fit in the active site. Even when there is incorrect addition , which happens in the case of   one in every 104 to 105 nucleotides ,Pol I will be there to fix this error in DNA replication using its selective method of active discrimination.

Effect of mutation in the 5' to 3 ' exonuclease activity of DNA polymerase I :

It will modify the 5' to 3 ' exonuclease activity of DNA polymerase I which will affect the proof reading capacity of the enzyme.

6) Function of Initiator protein:

The initiator ptotein that activates the initiation of DNA replication in bacteria is the DnaA which promotes the unwinding of DNA at oriC. The onset of the initiation phase of DNA replication is determined by the concentration of DnaA. Accumulation of DnaA occurs during growth and this triggers the initiation of replication.

Effect of mutation :

A mutation on Dna A protein makes it refractory to negative regulation.

7)HELICASE

Function of Helicase :

Enzymes that bind and may even remodel nucleic acid or nucleic acid protein complexes are known as Helicases . There are DNA and RNA helicases. DNA helicases are essential during DNA replication because they separate double-stranded DNA into single strands allowing each strand to be copied. During DNA replication, at positions called origins , DNA helicases unwind DNA , where synthesis will be initiated. DNA helicase continues to unwind the DNA forming a structure called the replication fork. It is named so because of its forked appearance of the two strands of DNA as they are unzipped apart. The process of breaking the hydrogen bonds between the nucleotide base pairs in double-stranded DNA requires energy. To break the bonds, helicases uses ATP molecule which are called the energy currency of the cell. DNA helicases also function in other cellular processes where double-stranded DNA must be separated, including DNA repair and transcription.

Effect of Mutation on helicases :

Analytical gel filtration showed that in the presence of an oligonucleotide, wild-type helicases form dimers whereas the mutant helicases remain mostly monomeric. This affects the helicase activity of separating the double strand into single strands.

8) Single stranded DNA binding protein:

It binds to single-stranded DNA rather than RNA or double-stranded DNA. Single-stranded DNA-binding proteins (SSB) have high affinity to single-stranded (ss) DNA and participate in DNA replication, recombination, and repair as accessory protein . These play a role in separating DNA strand during replication and prevent ssDNA from re-forming into a double helix.

Effect of Mutation:

A mutation in the Escherichia coli gene for single-strand binding protein results in temperature-sensitive deoxyribonucleic acid replication.

9) CLAMP PROTEIN:

A DNA clamp, also known as a sliding clamp, is a proteinfold that serves as a processivity-promoting factor in DNA replication. This protein binds DNA polymerase and prevents this enzyme from dissociating from the template DNA strand, as a critical component of the DNA polymerase III holoenzyme,

Effect of mutation:

dnaN5 allele of Bacillus subtilis encodes a mutant form of clamp containing the G73R substitution. Cells with the dnaN5 allele are temperature sensitive for growth due to a defect in DNA replication at 49°C, and they show an increase in mutation frequency caused by a partial defect in MMR at permissive temperatures.

10) Topoisomerases :

Function:

Enzymes that participate in the overwinding or underwinding of DNA are called as Topoisomerase. Topoisomerases are enzymes that participate in the overwinding or underwinding of DNA. The winding problem of DNA arises due to the intertwined nature of its double-helical structure. During DNA replication and transcription, DNA becomes overwound ahead of a replication fork. . The winding problem of DNA arises due to the intertwined nature of its double-helical structure. During DNA replication and transcription, DNA becomes overwound ahead of a replication fork.

Effect of mutation:

Resistance to the fluoroquinolones in salmonellae has mainly been attributed to mutations in the gyrA gene.

Polymerase I is able to actively discriminate

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