By Dr Ananya Mandal, MD
DNA is a polymer that lies within the nucleus of all cells. DNA interacts with numerous proteins that perform their functions in conjunction with DNA.
Some proteins that come in contact with DNA include several DNA modifying enzymes and some binding proteins.
DNA modifying enzymes
These include Methylases, Polymerases, Nucleases, Ligases, Kinase and Phosphatases. Each of these enzymes have specific function and activity. Some of the may be outlined as follows:
There are a variety of DNA methylases. These help in recognition of sequences on the DNA and their sequences are the same as endonucleases.
For example, an enzyme EcoR1 methylase recognizes and methylates at the sequence "GAATTC" on the DNA. These help in the transfer of a methyl group from S-adenosylmethionine (SAM) to a specific base in the recognition sequence. SAM here is important for the methylation reaction.
DNA is methylated to protect it against breakage and the action of related restriction endonucleases.
Some methylases have small specificity. For example Sss I methylase will methylate cytosine residues in the sequence 5' … CG … 3'. This means the methylated DNA will be protected from a wide variety of restriction endonucleases since the enzyme binds to several sites.
Some restriction endonucleases again only cut DNA at their recognition sites if the DNA is methylated for example Dpn I and some restriction endonucleases will cut both methylated and non-methylated DNA at their recognition sequences (e.g. BamH I).
There are a wide variety of polymerases that are present in nature and also synthesized commercially for use in DNA technology and laboratories working with DNA.
All DNA polymerases share two general characteristics:
- they add nucleotides to the 3'-OH end of a primer
- they place the sequence of nucleotides in the nascent or newly forming polynucleotide as per the template
The polymerase thus, helps in formation of the new polypeptide chain.
The polymerase can contain exonuclease activity. This exonuclease activity can proceed either in the 5'->3'direction, or in the 3'->5' direction.
Exonuclease activity in the 3'->5' direction allows the polymerase to correct a mistake if it incorporates an incorrect nucleotide. This is called “error correction activity”. It can also slowly degrade the 3' end of the primer.
Further Exonuclease activity in the 5'->3' direction will allow it to degrade any other hybridized primer it may encounter. This may also physically displace the obstructing primers.
Polymerases are used in several functions. Some polymerases are stable at high temperatures and are called thermostable polymerases. This has allowed the development of the "Polymerase Chain Reaction" technique (PCR), which has had a profound impact on modern Biotechnology. The polymerase reaction is terminated by incorporation of dideoxy bases (i.e. no hydroxyl groups on either the 2' or 3' carbon of the ribose sugar).
There are several nucleases. One of these is the Nuclease BAL-31. This is an exonuclease that starts at the termini and works inward that can degrade both 3' and 5' termini of double stranded DNA. This does not make internal cleavages ("nicks") within the DNA.
The degradation is not simultaneous and both ends are not similarly blunted. Such "ragged" ends can be made blunt by filling in and chewing back by a suitable polymerase (e.g. T4 DNA polymerase).
Another nuclease is the Exonuclease III. This catalyzes the stepwise removal of nucleotides from the 3' hydroxyl termini of duplex DNA. The enzyme attacks the 3' hydroxyl at duplex DNA with blunt ends. Since duplex DNA is required, the enzyme will not digest the 3' end of duplex DNA where the termini are 3' overhangs.
These are enzymes that catalyse the formation of a phosphodiester bond between 5' phosphate and 3' hydroxyl termini of nucleotides (potentially RNA or DNA depending on the ligase).
Ligases require either rATP or NAD+ as a cofactor, and this contrasts with restriction endonucleases. They are opposite of restriction endonucleases.
Some ligases include T4 DNA ligase that comes from a bacteria bacteriophage T4. It ligates or ties up ends of duplex DNA or RNA. This enzyme will join blunt-end termini. This enzyme will also repair single stranded nicks in duplex DNA, RNA or DNA/RNA duplexes.
Taq DNA ligase catalyzes a phosphodiester bond between two adjacent oligonucleotides which are hybridized to a complementary DNA strand. The enzyme is active at relatively high temperatures (45 - 65 °C) and requires NAD+ as a cofactor.
Other ligases include T4 RNA ligase and DNA ligases from E. coli.
T4 polynucleotide kinase
These catalyse the transfer and exchange of a phosphate group from rATP (adenine ribose triphosphate nucleotide) to the 5' hydroxyl terminus of double stranded and single stranded DNA or RNA, and nucleoside 3' monophosphates. The enzyme will also remove 3' phosphoryl groups.
Calf intestinal phosphatase (CIP)
These enzymes help in the removal of 5' phosphate groups from RNA, DNA and ribo- and deoxyribo- nucleoside triphosphates (e.g. ATP, rATP).
DNA binding proteins
There are several proteins that bind to specific sites in the genome to regulate genome expression and maintenance. Some of these include transcription activators that bind to specific promoter sequences and form chromatin modifying complexes that initiates RNA synthesis.
The reprogramming of gene expression also occurs when the cells progresses along the cell cycle or when cells sense changes in their environment. This is brought about by changes in the DNA binding status of transcriptional activators.
When DNA replicates, many different proteins and enzymes work together to accomplish the following steps:
- The two parent DNA strands are unwound or uncoiled with the help of DNA helicases. These are enzymes.
- Single stranded DNA binding proteins attach to the unwound strands, preventing them from winding or coiling back together.
- The strands are held in position, binding to DNA polymerase, which catalyzes the elongation of the leading and lagging strands.
- While the DNA polymerase on the leading strand can operate in a continuous fashion, RNA primer is needed repeatedly on the lagging strand to facilitate synthesis of Okazaki fragments.
- DNA primase, which is one of several polypeptides bound together in a group called primosomes, helps to build the primer.
- Finally, each new Okazaki fragment is attached to the completed portion of the lagging strand in a reaction catalyzed by DNA ligase.
Reviewed by April Cashin-Garbutt, BA Hons (Cantab)