Problem : What dictates which nucleoside triphosphate will be added next to a growing DNA chain?
The chemical addition of nucleotides to a growing DNA chain is dictated by the parent strand that is being copied. A nucleoside triphosphate with a complementary base to the one on the parent strand will be added to a growing chain.
Problem : What side group on a nucleoside triphosphate (building block of DNA) is responsible for mediating the addition of the next nucleotide?
The addition of nucleotides occurs through a nucleophilic attack by the 3' –OH on the deoxyribose sugar group of a nucleotide located at the end of a growing DNA chain.
Problem : What provides the energy for the addition of nucleotides to a growing DNA chain during replication?
During the addition reaction, a pyrophosphate group is released from the nucleoside triphosphate being added. The pyrophosphate's subsequent hydrolysis provides the energy that drives the addition reaction.
Problem : True or False. DNA replication occurs in the 5' to 3' direction because 3' to 5' replication is chemically impossible. Explain your answer.
False. While DNA replication does occur in the 5' to 3' direction, the reason is not because 3' to 5' replication is chemically impossible. 3' to 5' replication can in principle occur. The 3' –OH of the incoming nucleotide instead of the nucleotide attached to the end of the growing chain would be the attacking group.
Problem : Why is lagging strand replication more complicated than leading strand replication?
Lagging strand synthesis is more complex because of the requirement for DNA replication to take place in the 5' to 3' direction. Since parent strands are oriented in an anti-parallel fashion, one strand is oriented in the 5' to 3' direction, while the other strand is oriented in the 3' to 5' direction. Synthesis on the strand that is oriented in the 3' to 5' (leading strand) direction can occur easily because replication simply begins at the 3' end (synthesizing the 5' end of the daughter strand) and continues along with the replication fork. Synthesis on the strand that is oriented in the 5' to 3' direction (lagging strand) can not follow the direction of the replication fork because that would lead to 3' to 5' synthesis of the daughter strand. Instead synthesis must occur in small segments to preserve the proper synthesis direction.
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DNA polymerases are the enzymes that replicate DNA in living cells. They do this by adding individual nucleotides to the 3-prime hydroxl group of a strand of DNA. The process uses a complementary, single strand of DNA as a template.
The energy required to drive the reaction comes from cutting high energy phosphate bonds on the nucleotide-triphosphate's used as the source of the nucleotides needed in the reaction.
The illustration above highlights important aspects of the reaction.
DNA polymerases can not create new strands of DNA. They only synthesis double stranded DNA from single stranded DNA. The starting point is a a stretch of single stranded DNA which is double stranded for at least part of its length. In the polymerase chain reaction the double stranded stretch is created by attaching short DNA primers. In living cells, RNA primers are used.
DNA polymerase uses the bases of the longer strand as a template. During strand elongation, two phosphates are cleaved from the incoming nucleotide triphosphate and the resulting nucleotide monophosphate is added to the DNA strand. This results in the:
- Formation of a phosphodiester bond between the phosphate attached to the 5' carbon of the incoming nucleotide and the hydroxyl group on the trailing 3' carbon
- Release of a pyrophosphate molecule
- Extension of the DNA polymer by one nucleotide
Removing two phosphates from the incoming nucleotide and bonding the remaining phosphate to the oxygen on the 3' carbon of the existing strand maintains the repeating sugar-phosphate-sugar-phosphate pattern that makes up the backbone of each DNA polymer.
Orientation of the strand is important. Dependence on energy from the phosphates linked to the 5-prime carbon of the incoming nucleotides means that DNA polymerase can only extend DNA strands by adding nucleotides to the 3-prime end of a DNA strand.
Test your understanding of the concepts covered by this illustration with the DNA Polymerase concept questions.
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