Imagine the genome as a massive highway construction project. Thousands of crews are paving road at the same time, each working on its own short stretch. Each crew represents a replication fork. Under normal circumstances, if one crew encounters a problem--a cracked pipe or a large rock in the path of a line--they handle it locally. They pause briefly, clear the obstruction, and continue paving. Cells do something similar. When a replication fork encounters a lesion, local me

Let's reset the scene. A replication fork has stalled. The polymerase cannot move forward due to a lesion that it has reached that hasn't been repaired. If the lesion isn't bypassed by TLS or re-priming in the first 20-30 minutes after RPA-single stranded DNA detection, the fork risks collapsing. The cell's solution to this is surprisingly mechanical: it literally pushes the fork backward. This maneuver, called fork reversal , moves the lesion away from the polymerase and pla

When a polymerase stalls at a lesion, the fork enters a state called fork protection . Within minutes the cell detects RPA-coated single-stranded DNA, stabilizes the replisome, and protects the exposed DNA while it attempts to repair or bypass the lesion. Fork protection is the cell’s first response to a stalled DNA polymerase. The second response, if the lesion isn't successfully bypassed, is fork reversal. This is a more drastic structural rearrangement. Fork reversal repos

In the previous chapter, I introduced the two major pathways that repair DNA lesions: base excision repair (BER) and nucleotide excision repair (NER). Both operate throughout the genome and the cell cycle. But repair does not always occur before a replication fork arrives. Sometimes a lesion escapes these repair systems and remains in the DNA when S phase begins. When that happens, the replication machinery will eventually encounter it. What happens next depends on the size o

In addition to DNA polymerase errors, the human genome — even in a perfectly healthy cell — is under intense assault. The threats come from within and without. Endogenous chemicals generated by normal metabolism react with DNA. Exogenous agents from the environment — sunlight, pollutants, cigarette smoke, industrial chemicals — do the same. Together they alter DNA’s chemistry, nick its backbone, and distort its double helical structure. Replication errors are mistakes made wh

Surprisingly, the most common DNA polymerase error is not a mismatch at all. It's the insertion of a ribonucleotide, an RNA monomer, into a growing chain instead of a DNA monomer. When that occurs, the bases pair correctly. But the small chemical hydroxyl (-OH) group on the ribose sugar alters the structure of the DNA backbone and makes it more prone to cleavage, increasing the risk of strand breaks if not removed. DNA vs RNA . The sugar portion in a ribonucleotide is a ribos

DNA replication is astonishingly accurate — but not perfect. Let me quantify that. Before proofreading, replicative polymerases misincorporate roughly once every 100,000 to one million nucleotides. That’s too many. One misincorporation every 100,000 nucleotides would produce tens of thousands of errors in a replicated human genome. In reality, replicated human genomes contain only a handful. Clearly, additional mechanisms must be at work. Cells thus deploy repair pathways to

As the CMG helicase moves along the double helix, propelling the replisome forward and separating the DNA into leading and lagging strands, it creates a physical problem. DNA cannot simply be pulled apart without consequence. The act of unwinding one region of the double helix necessarily affects the regions ahead of it. This is often referred to as the “winding problem.” Let me explain with a human-scale analogy. Imagine two ropes wound tightly around each other like a doubl

DNA replication has a directional problem. The molecular machines that build DNA — the DNA polymerases — can add new nucleotides in only one direction: 5′ to 3′. That rule is absolute, dictated by the chemistry of the reaction itself. But the two strands of the DNA double helix run in opposite directions. They're antiparallel. So what happens when the replication fork moves forward? One strand can be copied smoothly and continuously. The other cannot. The second strand — th

Let's start this chapter off with two fundamental facts: one about DNA and the other about DNA polymerases. Fact one. Recall that the two DNA strands in a double helix are oriented in an antiparallel fashion. They run in opposite directions like the lanes of a two-lane highway. Scientists describe the orientation of a strand as either “5’-to-3’” (five prime to three prime) or “3’-to-5’” (three prime to five prime) based on the position of the sugars in the DNA backbone. Fact

Inside every dividing cell, rings of protein race along DNA. Some pry apart the double helix, strand by strand. Others snap into place to keep the copying machinery from slipping off as it works. Two of the most important of these molecular machines are the CMG helicase and the PCNA sliding clamp. The helicase acts as both engine and strand separator. It leads the replisome forward along the double helix while simultaneously splitting apart the two intertwined strands so they

17. Assembling the Replisome Our cell has now entered S phase. Across its genome, tens of thousands of starting points, or replication origins, have been prepared in advance. Each one has been “licensed”—approved for use. Replication origin licensing is based on the presence of two MCM protein complexes lined up tail-to-tail at the origin in question. For the time being, they are inert. Their transformation into CMG helicases (the protein complexes that unwind the double heli

As we kick off the focus of this book—a deep dive into human genome replication—we’ll see that the process involves many different proteins operating in an extremely well-orchestrated and interconnected manner. Tight regulation of the process is necessary to ensure the genome is replicated once and only once and with extremely high accuracy.     Although the copying of the human genome occurs during S-phase of the cell cycle, the events described in this post--that is, the at

If someone had wanted a book in the 15th century it would have had to have been copied by hand from an existing one. The scribe would have started at page one and proceeded page by page until the last page was, as they say, “in the books.”   A human genome is like a book. So, to copy it, one might imagine the cell would use the same straightforward approach: start at one end of each of the 46 chromosomes and progress to the other. Unfortunately, that wouldn’t work. Human cell

Having been just reviewed the cell cycle we now focus on the fourth step of that process, mitosis, where the two genomes are pulled into their new cells. This is an amazing feat of cellular choreography that I liken to a line dance. Let's take a quick look at the entire mitosis process from start to finish before covering each step in more detail. The entry point is the very end of interphase--that is, the combination of phases G1, S, and G2. The genome has been copied and al

Despite the title of this chapter, most of the time, most of your cells are just doing their jobs. They're not dividing. Nerve cells are transmitting electrochemical signals. Heart muscle cells are contracting rhythmically. Rod and cone cells in the retina are detecting the words you’re reading. But it's also true that your body is constantly renewing itself. Every day it replaces about 300 billion cells (1). That’s an enormous number--but it still represents less than one pe

Each time a cell divides, it creates a copy of both its genomes for the two daughter cells. It does so with extremely high accuracy (or "fidelity" in molecular biology parlance). Human cells copy their genomes with 99.99999999% accuracy. That equates to one single error per every 100 million nucleotides! Stop and think about that! In this post, we're going to review eight reasons why replicating the human genome is so challenging. Of course, the cell overcomes these challen

In this post I present a perfect example of ATP-driven protein motor movement. ATP is a frequently employed driver of protein movement in all living cells. The goal here is to absorb a general concept that we'll see several more times as we learn about genome replication. Specifically, today, we'll look at myosin , a protein complex found in skeletal muscle cells. We'll see what, exactly, makes myosin move and how minute movements at the molecular level in muscle cells lead t

A cell is a dynamic place. Enzymes are speeding up chemical reactions. DNA, RNA, and proteins are being synthesized. Molecular motors are carrying organelles around the cell. Enzymes are repairing DNA lesions. The cell membrane is selectively letting specific molecules and ions in and out of the cell. The list goes on. It's a bustling environment! But what is the energy source fueling all this activity? One good answer is "food." The food we eat consists of various kinds of

Two human genomes adding up to 6 billion base pairs are contained in the nucleus of every cell in your body. Different kinds of cells use different genes. That depends on the cell’s job. But every cell contains the whole genome, or every human gene.   One of the cell's challenges is that the genome is very large but its nucleus is very small. If we could stretch out the human genome--in other words line up the 6 billion base pairs end to end--it would stretch nearly 6 feet! Y