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Molecular Biology Review

DNA Replication

Replication is the process by which DNA is synthetized in vivo, and requires a pre-existing DNA molecule as template, a primer, a replication origin, DNA polymerase, deoxynucleotides (dNTPs), and ligase. It starts with the unwinding of a DNA template and separation of the strands forming a structure known as a replication fork. Each strand can then serve as a template. A shot RNA sequence serves as a primer by annealing to the replication origin, for example, to ori C in the E. coli circular chromosome. Eukaryotes have many replication origins because they have more than one chromosome. DNA polymerase recognizes and binds to the replication origin, the starts catalyzing the addition of nucleotides to the primer.

DNA replication always moves from the 5' end to the 3' end of the newly synthezized molecule. Because one of the template strands is not completely unwond, replication of that strand occurs in pieces, adding new primers as the template unwinds. The DNA fragments produced are known as Okazaki fragments and are eventually joined by ligase. The new DNA strand synthetized in fragments is known as the lagging strand, while the strand synthetized continuously is known as the leading strand.

Usually, a molecule of DNA polymerase does not compleately replicates a strand, but "jumps around" from template to template. This phenomenom is known as distrivuive synthesis. Some isozymes stay on one tamplate longer than others, thus are more efficient or have better processivity.

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Properties of DNA

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Degradation Enzymes

Nucleases cut at either side of the phosphodiester bond of polynucleotides:

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Some nucleases cut both DNA and RNA, while others cut only DNA (DNases) or only RNA (RNases). Nucleases that cut only at or near the end of a polynucleotide are known as exonucleases, while endonucleases only cut at interior base pairs. Some commonly used nucleases are Bal31, ExoIII, S1, mung bean nuclease, DNase 1, RNase A and RNase H.

Bal31 is obtained from Alteromones espejiana and cuts polynucleotides. It acts as a single strand endonuclease and a double strand exonuclease in the prescence of Ca2+. Bal31 is commonly used to make division, for example to isolate the sequence of interest and make blunt ends by removing unwanted base pairs.

ExoIII recognizes double stranded polynucleotides but cut only one strand. It is a 3'-to-5' exonuclease that leaves a 5' overhang, but cannot cut if there is a 3' overhang. ExoIII is used as Bal31, then another enzyme is used to remove the 5' overhang.

S1 nuclease from Aspergillus oryzae cuts polynucleotides as either an endonuclease or an exonuclease in the prescence of Zn2+. It is single strand specific, but will cut a double strand that is already nicked. Mung bean nuclease is identical to S1 except it will not cut nicked double stranded DNA.

DNase 1 is a DNA endonuclease. If Mg2+ is present, DNase 1 will recognize double stranded DNA but cut only one strand. If Mn2+ is present, it will cut both strands.

RNase H cuts only the RNA in RNA/DNA hybrid helices. RNase A is specific to single stranded RNA (cannot cut at hairpins), cutting in front of C or U.

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Restriction Endonucleases

Restriction endunucleases originate from one of two enzymatic systems in bacteria: hsd (host specificity for DNA) or MDRS (methylation-dependent restriction system).

The hsd genes confere protection to bacteria against bacteriophages (viruses that infect bacteria). Different strains of bacteria have different hsd genes that produce restriction endonucleases and methylases. The methylases modify bacterial DNA by methylating at either adenine or cytocine. The restriction endonucleases degrade DNA if not methylated, protecting the bacteria from non-methylated foreign DNA.

But some bacteriophages may also methylate their DNA, thus using hsd bacteria may be able to protect themselves only against bacteriophages with methylation patterns different to their own. For example, if the bacteriophage lambda kappa infects E. coli kappa, the bacteria will not be able to fight the infection because both organisms have the same methylation pattern. On the other hand, restriction endonucleases from E. coli beta should be able to degrade lambda kappa.

The MDRS enzymes cut methylated DNA only, thus E. coli does not methylate its own genomic DNA at the MDRS recognition sites, thus the MDRS system does not produce methylases as hsd do. MDRS genes may be of two types: modified cytosine restriction (MCR) or methyladenine recognition restriction (MAR).

Restriction endonucleases degrade bacteriophage DNA by cutting at specific double-stranded DNA sequences (they do not cut single stranded DNA), for example:

5' ----- GAATTC ----- 3' 3' ----- CTTAAG ----- 5'

EcoRI

5' ----- G               AATTC ----- 3' 3' ----- CTTAA               G ----- 5'

An enzyme like EcoRI (from E. coli) leaves "sticky ends" with a 5' overhang. Other enzymes may leave either a 3' ovehang or blunt ends:

There are three types of restriction endonucleases. The most commonly used Type II restriction endonucleases are simple proteins that require only Mg2+ and generally cut at a palindromic host specific recognition site. Other types of restriction endonuclease require Mg2+, AdoMet and/or ATP and may cut many base pairs away from the recognition site, sometimes at random.

Isoschizomers are restriction endonucleases that recognize the same sequence and may cut at the same or different bases. If they cut at different bases, they are called neoschizomers. Isoschizomers may be affected differently by methylation. For example: MboI, Sau3A and DpnI are isochizomers (DpnI is a neoschizomer) that recognize 5'--GATC--3'. MboI will cut only the unmethylated sequence, Sau3A will cut either the one-strand methylated or unmethylated sequence, and DpnI will cut only if the sequence is metylated in both strands.

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Synthesis and Modification Enzymes

Synthetic enzymes include DNA polymerases and RNA polymerases. There are many isozymes of DNA polymerase, some commonly use in biolotechnology are E. coli, T7, T4, Taq and reverse transcriptase.

E. coli DNA polymerase has two subunits: a 76 kD 5' to 3' polymerase and a 35 kD 3' to 5' exonuclease. The exonuclease only cut one strand of the double stranded DNA, and in the bacteria is used to proofread a newly sinthezized DNA strand. The polymerase does not recognize individual nucleotides, it just ads any nucleoride, and it is up to the exonuclease to identify the incorrect pairings and remove the wrong nucleotide just added. The 3'-to-5' exonuclease identidies the wrong bases by reconizing that the H bonds are not forming. Such proofreading will continue until the correct base is in place.

T7 DNA polymerase, also known as sequenace (?), is made of two subunits: 5' to 3' polymerase and 3' to 5' exonuclease. Although similar to E. coli, T7 has better processivity, therefore it is often used for manual DNA sequencing.

T4 DNA polymerase also has the 5' to 3' polymerase and 3' to 5' exonuclease activities, but its exonuclease is single-strand specific. It is mostly used to remove 3' overhangs.

Taq DNA polymerase comes from Thermus aquaticus, a bacteria that lives in hot springs. It is used for reactions that need to occur at very high temperatures, like the polymerase chain reaction (PCR).

There are also many RNA polymerase isozymes. Prokaryotic isozymes like E. coli RNA polymerase make mRNA, tRNA and rRNA. Other commonly used RNA polymerases includ Sp6 (from Salmonella), T7 and T3. Eukaryotic have different RNA polymerases that make the divers kinds of RNA:

• RNA Polymerase I: 18s, 28s and 5.8 s rRNA
• RNA Polymerase II: mRNA and snRNA
• RNA Polymerase III: tRNA and 5s rRNA

Modification enzymes include methylases, lagases, alkaline phosphatases and kinases. As already discussed, methylases add a methyl group at either adenine or cytocine:

Ligases join two pieces of DNA together by forming phosphodiester bonds:

Phosphatases remove the 5' phosphate of polynucleotides, while kinase adds a phosphate to an already dephosphorilated DNA 5' end.

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